Factor VIII zwitterionic polymer conjugates

ABSTRACT

The present invention provides multi-armed high MW polymers containing hydrophilic groups conjugated to Factor VIII, and methods of preparing such polymers.

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

Any and all applications for which a foreign or domestic priority claimis identified in the Application Data Sheet as filed with the presentapplication are hereby incorporated by reference under 37 CFR 1.57.

REFERENCE TO SEQUENCE LISTING

A Sequence Listing submitted as an ASCII text file via EFS-Web is herebyincorporated by reference in accordance with 35 U.S.C. § 1.52(e). Thename of the ASCII text file for the Sequence Listing isKDIAK008C1SEQLIST.txt, the date of creation of the ASCII text file isApr. 29, 2020, and the size of the ASCII text file is 19,707 bytes.

BACKGROUND

Hemophilia A is a hereditary blood coagulation disorder caused bydeficits of or mutations in Factor VIII (FVIII). Factor VIII is acrucial component in the intrinsic blood coagulation pathway.Deficiencies in Factor VIII cause increased bleeding. Hemophilia A is Xlinked and is observed in males at about 1 in 5000.

Hemophilia A patients are currently treated by intravenousadministration of full length recombinant human FVIII. Treatment may beprophylactic or as necessitated in response to an injury causingbleeding. The half-life of Factor VIII in humans is relatively short,typically on the order of 11 hours. Hence, frequent dosages on the orderof three times a week are required for effective treatment. However,such frequent dosing, typically via infusion, is undesirable,necessitating frequent visits to a clinic or other healthcare provider.Moreover, such frequent dosing may diminish patient compliance withprescribed dosing regimes.

Another drawback with current Factor VIII treatment is that some 25 to30% of Factor VIII treated patients develop antibodies to FVIII.Patients with high levels of circulating anti-Factor VIII antibodiescannot be successfully treated with current Factor VIII therapeutics.Such patients require a more expensive treatment regime involving FactorVila and immune tolerance therapy.

The efficacy of a therapeutic agent may be enhanced by improving itsbioavailability and pharmacokinetic properties. One approach toimproving bioavailability has been PEGylation. PEGylation involves theaddition of polyethylene glycol chains to a drug, typically a protein. Areduction in immunogenicity or antigenicity, increased half-life,increased solubility, decreased clearance by the kidney and decreasedenzymatic degradation have been attributed to PEG conjugates. As aresult of these attributes, it has been reported that PEG conjugates ofcertain biologically active agents sometimes require less frequentdosing and may permit the use of less of the active agent to achieve atherapeutic endpoint. Less frequent dosing is generally desirablebecause it reduces the overall number of injections, which can bepainful and which require inconvenient visits to healthcareprofessionals.

Although some success has been achieved with PEG conjugation,“PEGylation” of biologically active agents remains a challenge. As drugdevelopers progress beyond very potent agonistic proteins such aserythropoietin and the various interferons, potential benefits of thePEG hydrophilic polymer in increased solubility, stability andbioavailability do not sufficiently compensate for increased viscosityand immunogenicity.

PEGylation of FVIII has not been observed to significantly increase thehalf-life of the conjugate in vivo.

Thus there remains a need for FVIII drugs with increased in vivohalf-life while retaining sufficient biological activity.

BRIEF SUMMARY OF THE CLAIMED INVENTION

The invention provides a conjugate comprising recombinant FVIII (rFVIII)and a zwitterionic polymer wherein the polymer comprises one or moremonomer units and wherein at least one monomer unit comprises azwitterionic group. Optionally, the zwitterionic group comprisesphosphorylcholine. Optionally, the monomer comprises2-(acryloyloxyethyl)-2′-(trimethylammoniumethyl) phosphate. Optionally,the monomer comprises2-(methacryloyloxyethyl)-2′-(trimethylammoniumethyl) phosphate(HEMA-PC). The rFVIII may have a deletion of part or all of the B domainor may have an intact B-domain. Optionally, the polymer has 3 or morearms. Optionally, the polymer has 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12arms. Optionally, the polymer has 3, 6 or 9 arms, preferably the polymerhas 9 arms.

Some conjugates are such that the polymer portion has a peak molecularweight of between 300,000 and 1,750,000 daltons including the rFVIII.Some conjugates have a polymer portion with a peak molecular weightbetween 500,000 and 1,000,000 daltons.

Some conjugates have a polymer portion with a peak molecular weightbetween 600.000 to 800,000 daltons.

In some conjugates the rFVIII is covalently bonded to the polymer. Insome conjugates, the polymer is covalently bonded to at least one of anamino group, a hydroxyl group, a sulfhydryl group and a carboxyl groupof rFVIII. In some conjugates, the sulfhydryl group is from a cysteineresidue in rFVIII. In some conjugates, the cysteine residue is arecombinant cysteine residue. In some conjugates, the recombinantcysteine residue is selected from the group consisting of Y81C, F129C,K377C, H378C, K422C, Q468C, L491C, L504C, K556C, K570C, D1795C, Q1796C,R1803C, K1804C, K1808C, K1810C, T1821C, K813C, N1864C, T1911C, N2118C,Q2091C, F2093C, and Q2284C, wherein residues are numbered fromcorresponding residues in SEQ ID NO:1 (FIG. 1 ), when the recombinantFactor VIII is maximally aligned with SEQ ID NO:1. In some conjugates,the cysteine residue is naturally present in rFVIII. In some conjugates,the cysteine residue is in the B domain. In some conjugates, thecysteine residue is selected from the group consisting of 1293C, 1373C,1604C and 1636C, preferably 1604C or 1636C.

A preferred conjugate has a polymer portion with a peak molecular weightof between 100.000 and 1,500,000, more preferably 500,000 to 1,000,000daltons or 600,000 to 850,000 daltons including rFVIII and 3, 6, or 9arms, preferably 9 arms.

The invention further provides a conjugate comprising recombinant FVIII(rFVIII) with at least a portion of a B domain and a zwitterionicpolymer wherein the polymer comprises one or more monomer units andwherein at least one monomer unit comprises a zwitterionic group and thepolymer is conjugated to the rFVIII via a cysteine residue in theB-domain, wherein one branched polymer is conjugated per molecule ofrFVIII. Optionally, the polymer is a branched polymer, optionally with 9branches. Optionally, the polymer is conjugated via a cysteine residuethat is one of the two C-terminal most cysteine residues in the portionof the B domain. Optionally, the conjugate has an in vivo half-life inhumans of at least 20 hours.

The invention further provides a composition comprising molecules of aconjugate comprising recombinant FVIII (rFVIII) including a light chainand a heavy chain including at least a portion of a B domain and azwitterionic polymer wherein the polymer comprises one or more monomerunits and wherein at least one monomer unit comprises a zwitterionicgroup and the polymer is conjugated to the rFVIII via a cysteine residuein the B-domain, wherein at least 80, 90, 95 or 99% of molecules of theconjugate in the composition have the same portion of the B domain andone polymer is conjugated per molecule of rFVIII. Optionally, thepolymer is branched, optionally with 9 branches. Optionally, the heavychain includes at least residues 1-1604 of SEQ ID NO:1 or at leastresidues 1-1636 of SEQ ID NO: 1, or at least residues 1-1648 of SEQ IDNO:1. Optionally, the heavy chain consists of residues 1-1648 of SEQ IDNO:1. Optionally, the at least a portion of the B domain is an intact Bdomain. Optionally, the polymer is conjugated via a cysteine that is oneof the two most C-terminal cysteines in the B domain.

The invention further provides a pharmaceutical composition comprising aconjugate as described above.

The invention further provides a method of treating hemophiliacomprising administering a therapeutically effective amount of aconjugate as described above to a subject suffering from hemophilia.

The invention further provides a method of prophylaxis of a subject withhemophilia, comprising administering a therapeutically effective amountof a conjugate or composition of any preceding claim to the subject withhemophilia at a time when the subject is not known to be bleedingexternally or internally, wherein the conjugate persists in the blood soas to promote clotting after subsequent bleeding. Optionally, theconjugate or composition is administered no more frequently than once aweek. Optionally, the conjugate or composition is administered betweenweekly and monthly. Optionally, the subject has a trough level of FVIIIactivity of greater than >1%, 3%, or 5% of the mean FVIII activity incontrol subjects without hemophilia. Optionally, the subject hasdeveloped antibodies to FVIII from previous administration of FVIIIunconjugated to the polymer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the amino acid sequence of mature, human Factor VIII (SEQID NO:1).

FIG. 2 shows the domains of human Factor VIII and location of cysteineresidues (reproduced from Lenting et al. Blood 1998:92:3983-3996)

DETAILED DESCRIPTION I. General

The present invention provides high molecular weight (MW) polymershaving hydrophilic groups or zwitterions, such as phosphorylcholine.Also provided in accordance with the present invention are methods andnovel starting materials for making the high MW polymers. Also providedin accordance with the present invention are conjugates of the high MWpolymers and functional agent (as defined herein). International PatentApplication Nos. PCT/US20 11/032768 and PCT/US2007/005372 are herebyincorporated by reference for all purposes.

II. Definitions

“Polymer” refers to a series of monomer groups linked together. The highMW polymers are prepared from monomers that include, but are not limitedto, acrylates, methacrylates, acrylamides, methacrylamides, styrenes,vinyl-pyridine, vinyl-pyrrolidone and vinyl esters such as vinylacetate. Additional monomers are useful in the high MW polymers of thepresent invention. When two different monomers are used, the twomonomers are called “comonomers.” meaning that the different monomersare copolymerized to form a single polymer. The polymer can be linear orbranched. When the polymer is branched, each polymer chain is referredto as a “polymer arm.” The end of the polymer arm linked to theinitiator moiety is the proximal end, and the growing-chain end of thepolymer arm is the distal end. On the growing chain-end of the polymerarm, the polymer arm end group can be the radical scavenger, or anothergroup.

“Initiator” refers to a compound capable of initiating a polymerizationusing the monomers or comonomers of the present invention. Thepolymerization can be a conventional free radical polymerization orpreferably a controlled/“living” radical polymerization, such as AtomTransfer Radical Polymerization (ATRP), ReversibleAddition-Fragmentation-Termination (RAFT) polymerization or nitroxidemediated polymerization (NMP). The polymerization can be a “pseudo”controlled polymerization, such as degenerative transfer. When theinitiator is suitable for ATRP, it contains a labile bond which can behomolytically cleaved to form an initiator fragment, I, being a radicalcapable of initiating a radical polymerization, and a radical scavenger.I′, which reacts with the radical of the growing polymer chain toreversibly terminate the polymerization. The radical scavenger F istypically a halogen, but can also be an organic moiety, such as anitrile.

“Linker” refers to a chemical moiety that links two groups together. Thelinker can be cleavable or non-cleavable. Cleavable linkers can behydrolyzable, enzymatically cleavable, pH sensitive, photolabile, ordisulfide linkers, among others. Other linkers include homobifunctionaland heterobifunctional linkers. A “linking group” is a functional groupcapable of forming a covalent linkage consisting of one or more bonds toa bioactive agent. Nonlimiting examples include those illustrated inTable 1.

The term “reactive group” as used herein refers to a group that iscapable of reacting with another chemical group to form a covalent bond,i.e. is covalently reactive under suitable reaction conditions, andgenerally represents a point of attachment for another substance. Thereactive group is a moiety, such as maleimide or succinimidyl ester, onthe compounds of the present invention that is capable of chemicallyreacting with a functional group on a different compound to form acovalent linkage. Reactive groups generally include nucleophiles,electrophiles and photoactivatable groups.

“Functional agent” is defined to include a bioactive agent or adiagnostic agent. A “bioactive agent” is defined to include any agent,drug, compound, or mixture thereof that targets a specific biologicallocation (targeting agent) and/or provides some local or systemicphysiological or pharmacologic effect that can be demonstrated in vivoor in vitro. Non-limiting examples include drugs, vaccines, antibodies,antibody fragments, scFvs, diabodies, avimers, vitamins and cofactors,polysaccharides, carbohydrates, steroids, lipids, fats, proteins,peptides, polypeptides, nucleotides, oligonucleotides, polynucleotides,and nucleic acids (e.g., mRNA, tRNA, snRNA. RNAi, DNA, cDNA, antisenseconstructs, ribozymes, etc). A “diagnostic agent” is defined to includeany agent that enables the detection or imaging of a tissue or disease.Examples of diagnostic agents include, but are not limited to,radiolabels, fluorophores and dyes.

“Therapeutic protein” refers to peptides or proteins that include anamino acid sequence which in whole or in part makes up a drug and can beused in human or animal pharmaceutical applications. Numeroustherapeutic proteins are known including, without limitation, thosedisclosed herein.

“Phosphorylcholine,” also denoted as “PC,” refers to the following:

where * denotes the point of attachment. The phosphorylcholine is azwitterionic group and includes salts (such as inner salts), andprotonated and deprotonated forms thereof.

“Phosphorylcholine containing polymer” is a polymer that containsphosphorylcholine. “Zwitterion containing polymer” refers to a polymerthat contains a zwitterion.

Poly(acryloyloxyethyl phosphorylcholine) containing polymer refers to apolymer containing 2-(acryloyloxy)ethyl-2-(trimethylammonium)ethylphosphate as monomer.

Poly(methacryloyloxyethyl phosphorylcholine) containing polymer refersto a polymer containing2-(methacryloyloxy)ethyl-2-(trimethylammonium)ethyl phosphate asmonomer.

“Molecular weight” in the context of the polymer can be expressed aseither a number average molecular weight, or a weight average molecularweight or a peak molecular weight. Unless otherwise indicated, allreferences to molecular weight herein refer to the peak molecularweight. These molecular weight determinations, number average (Mn),weight average (Mw) and peak (Mp), can be measured using size exclusionchromatography or other liquid chromatography techniques. Other methodsfor measuring molecular weight values can also be used, such as the useof end-group analysis or the measurement of colligative properties(e.g., freezing-point depression, boiling-point elevation, or osmoticpressure) to determine number average molecular weight, or the use oflight scattering techniques, ultracentrifugation or viscometry todetermine weight average molecular weight. In a preferred embodiment ofthe present invention, the molecular weight is measured by SEC-MALS(size exclusion chromatography—multi angle light scattering). Thepolymeric reagents of the invention are typically polydisperse (i.e.,number average molecular weight and weight average molecular weight ofthe polymers are not equal), preferably possessing low polydispersityvalues of, for example, less than about 1.5, as judged by gel permeationchromatography. In other embodiments, the polydispersities (PDI) aremore preferably in the range of about 1.4 to about 1.2, still morepreferably less than about 1.15, and still more preferably less thanabout 1.10, yet still more preferably less than about 1.05, and mostpreferably less than about 1.03.

The phrase “a” or “an” entity as used herein refers to one or more ofthat entity; for example, a compound refers to one or more compounds orat least one compound. As such, the terms “a” (or “an”), “one or more”,and “at least one” can be used interchangeably herein.

“About” as used herein means variation one might see in measurementstaken among different instruments, samples, and sample preparations.

“Protected.” “protected form.” “protecting group” and “protective group”refer to the presence of a group (i.e., the protecting group) thatprevents or blocks reaction of a particular chemically reactivefunctional group in a molecule under certain reaction conditions.Protecting groups vary depending upon the type of chemically reactivegroup being protected as well as the reaction conditions to be employedand the presence of additional reactive or protecting groups in themolecule, if any. Suitable protecting groups include those such as foundin the treatise by Greene et al., “Protective Groups In OrganicSynthesis,” 3^(rd) Edition, John Wiley and Sons, Inc., New York, 1999.

“Alkyl” refers to a straight or branched, saturated, aliphatic radicalhaving the number of carbon atoms indicated. For example, Ci-C₆ alkylincludes, but is not limited to, methyl, ethyl, propyl, isopropyl,butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc.Other alkyl groups include, but are not limited to heptyl, octyl, nonyl,decyl, etc. Alkyl can include any number of carbons, such as 1-2, 1-3,1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 3-4, 3-5, 3-6,4-5, 4-6 and 5-6. The alkyl group is typically monovalent, but can bedivalent, such as when the alkyl group links two moieties together.

The term “lower” referred to above and hereinafter in connection withorganic radicals or compounds respectively defines a compound or radicalwhich can be branched or unbranched with up to and including 7,preferably up to and including 4 and (as unbranched) one or two carbonatoms.

“Alkylene” refers to an alkyl group, as defined above, linking at leasttwo other groups, i.e., a divalent hydrocarbon radical. The two moietieslinked to the alkylene can be linked to the same atom or different atomsof the alkylene. For instance, a straight chain alkylene can be thebivalent radical of —(CH₂)_(n), where n is 1, 2, 3, 4, 5 or 6. Alkylenegroups include, but are not limited to, methylene, ethylene, propylene,isopropylene, butylene, isobutylene, sec-butylene, pentylene andhexylene.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be a variety of groups selected from: —OR′, ═0,═NR′, ═N—OR′, —NR′R″, —SR′, -halogen, —SiR′R″R″′, —OC(0)R\ —C(0)R\—C0₂R′, —CONR′R″, —OC(0)NR′R″, —NR″C(0)R′, —NR′—C(0)NR″R″, —NR″C(0)₂R′,—NH—C(NH₂)═NH, —NR′C(NH₂)═NH, —NH—C(NH₂)═NR′, —S(0)R\ —S(0)₂R′,—S(0)₂NR′R″, —CN and —N0₂ in a number ranging from zero to (2m′+1),where m′ is the total number of carbon atoms in such radical. R′, R″ andR′″ each independently refer to hydrogen, unsubstituted (Ci-Cs)alkyl andheteroalkyl, unsubstituted aryl, aryl substituted with 1-3 halogens,unsubstituted alkyl, alkoxy or thioalkoxy groups, or aryl-(Ci-C4)alkylgroups. When R′ and R″ are attached to the same nitrogen atom, they canbe combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups such as haloalkyl (e.g., —CF and —CH₂CF₃) and acyl (e.g.,—C(0)CH₃, —C(0)CF₃, —C(0)CH₂OCH₃, and the like). Preferably, thesubstituted alkyl and heteroalkyl groups have from 1 to 4 substituents,more preferably 1, 2 or 3 substituents. Exceptions are those perhaloalkyl groups (e.g., pentafluoroethyl and the like) which are alsopreferred and contemplated by the present invention.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═0, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R″′, —OC(0)R\ —C(0)R\ —C0₂R′, —CONR′R″, —OC(0)NR′R″,—NR″C(0)R′, —NR′—C(0)NR″R″—NR″C(0)₂R′, —NR—C(NR′R″R″′)═NR″″,—NR—C(NR′R″)═NR″′, —S(0)R′, —S(0)₂R′, —S(0)₂NR′R″, —NRS0₂R′, —CN and—N0₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound of the inventionincludes more than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R′″ and R″″ groups when morethan one of these groups is present. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include,but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the abovediscussion of substituents, one of skill in the art will understand thatthe term “alkyl” is meant to include groups including carbon atoms boundto groups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(0)CH₃, —C(0)CF₃, —C(0)CH₂OCH₃, and thelike).

“Alkoxy” refers to alkyl group having an oxygen atom that eitherconnects the alkoxy group to the point of attachment or is linked to twocarbons of the alkoxy group. Alkoxy groups include, for example,methoxy, ethoxy, propoxy, iso-propoxy, butoxy, 2-butoxy, iso-butoxy,sec-butoxy, tert-butoxy, pentoxy, hexoxy, etc. The alkoxy groups can befurther substituted with a variety of substituents described within. Forexample, the alkoxy groups can be substituted with halogens to form a“halo-alkoxy” group.

“Carboxyalkyl” means an alkyl group (as defined herein) substituted witha carboxy group. The term “carboxycycloalkyl” means an cycloalkyl group(as defined herein) substituted with a carboxy group. The term alkoxyalkyl means an alkyl group (as defined herein) substituted with analkoxy group. The term “carboxy” employed herein refers to carboxylicacids and their esters.

“Haloalkyl” refers to alkyl as defined above where some or all of thehydrogen atoms are substituted with halogen atoms. Halogen (halo)preferably represents chloro or fluoro, but may also be bromo or iodo.For example, haloalkyl includes trifluoromethyl, fluoromethyl,1,2,3,4,5-pentafluoro-phenyl, etc. The term “perfluoro” defines acompound or radical which has all available hydrogens that are replacedwith fluorine. For example, perfluorophenyl refers to1,2,3,4,5-pentafluorophenyl, perfluoromethyl refers to1,1,1-trifluoromethyl, and perfluoromethoxy refers to1,1,1-trifluoromethoxy.

“Fluoro-substituted alkyl” refers to an alkyl group where one, some, orall hydrogen atoms have been replaced by fluorine.

“Cytokine” in the context of this invention is a member of a group ofprotein signaling molecules that may participate in cell-cellcommunication in immune and inflammatory responses. Cytokines aretypically small, water-soluble glycoproteins that have a mass of about8-35 kDa.

“Cycloalkyl” refers to a cyclic hydrocarbon group that contains fromabout 3 to 12, from 3 to 10, or from 3 to 7 endocyclic carbon atoms.Cycloalkyl groups include fused, bridged and spiro ring structures.

“Endocyclic” refers to an atom or group of atoms which comprise part ofa cyclic ring structure.

“Exocyclic” refers to an atom or group of atoms which are attached butdo not define the cyclic ring structure.

“Cyclic alkyl ether” refers to a 4 or 5 member cyclic alkyl group having3 or 4 endocyclic carbon atoms and 1 endocyclic oxygen or sulfur atom(e.g., oxetane, thietane, tetrahydrofuran, tetrahydrothiophene); or a 6to 7 member cyclic alkyl group having 1 or 2 endocyclic oxygen or sulfuratoms (e.g., tetrahydropyran, 1,3-dioxane, 1,4-dioxane,tetrahydrothiopyran, 1,3-dithiane, 1,4-dithiane, 1,4-oxathiane).

“Alkenyl” refers to either a straight chain or branched hydrocarbon of 2to 6 carbon atoms, having at least one double bond. Examples of alkenylgroups include, but are not limited to, vinyl, propenyl, isopropenyl,1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl,isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl,3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl,2,4-hexadienyl, or 1,3,5-hexatrienyl. Alkenyl groups can also have from2 to 3, 2 to 4, 2 to 5, 3 to 4, 3 to 5.3 to 6, 4 to 5, 4 to 6 and 5 to 6carbons. The alkenyl group is typically monovalent, but can be divalent,such as when the alkenyl group links two moieties together.

“Alkenylene” refers to an alkenyl group, as defined above, linking atleast two other groups, i.e., a divalent hydrocarbon radical. The twomoieties linked to the alkenylene can be linked to the same atom ordifferent atoms of the alkenylene. Alkenylene groups include, but arenot limited to, ethenylene, propenylene, isopropenylene, butenylene,isobutenylene, sec-butenylene, pentenylene and hexenylene.

“Alkynyl” refers to either a straight chain or branched hydrocarbon of 2to 6 carbon atoms, having at least one triple bond. Examples of alkynylgroups include, but are not limited to, acetylenyl, propynyl, 1-butynyl,2-butynyl, isobutynyl, sec-butynyl, butadiynyl, 1-pentynyl, 2-pentynyl,isopentynyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl,3-hexynyl, 1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl,2,4-hexadiynyl, or 1,3,5-hexatriynyl. Alkynyl groups can also have from2 to 3, 2 to 4, 2 to 5, 3 to 4, 3 to 5, 3 to 6, 4 to 5, 4 to 6 and 5 to6 carbons. The alkynyl group is typically monovalent, but can bedivalent, such as when the alkynyl group links two moieties together.

“Alkynylene” refers to an alkynyl group, as defined above, linking atleast two other groups, i.e., a divalent hydrocarbon radical. The twomoieties linked to the alkynylene can be linked to the same atom ordifferent atoms of the alkynylene. Alkynylene groups include, but arenot limited to, ethynylene, propynylene, butynylene, sec-butynylene,pentynylene and hexynylene.

“Cycloalkyl” refers to a saturated or partially unsaturated, monocyclic,fused bicyclic or bridged poly cyclic ring assembly containing from 3 to12 ring atoms, or the number of atoms indicated. Monocyclic ringsinclude, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,and cyclooctyl. Bicyclic and polycyclic rings include, for example,norbornane, decahydronaphthalene and adamantane. For example,c₃₋₈cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cyclooctyl, and norbornane.

“Cycloalkylene” refers to a cycloalkyl group, as defined above, linkingat least two other groups, i.e., a divalent hydrocarbon radical. The twomoieties linked to the cycloalkylene can be linked to the same atom ordifferent atoms of the cycloalkylene. Cycloalkylene groups include, butare not limited to, cyclopropylene, cyclobutylene, cyclopentylene,cyclohexylene, and cyclooctylene.

“Heterocycloalkyl” refers to a ring system having from 3 ring members toabout 20 ring members and from 1 to about 5 heteroatoms such as N, O andS. Additional heteroatoms can also be useful, including, but not limitedto, B, Al, Si and P. The heteroatoms can also be oxidized, such as, butnot limited to, —S(O)— and —S(0)₂. For example, heterocycle includes,but is not limited to, tetrahydrofuranyl, tetrahydrothiophenyl,morpholino, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl,pyrazolidinyl, pyrazolinyl, piperazinyl, piperidinyl, indolinyl,quinuclidinyl and 1,4-dioxa-8-aza-spiro[4.5]dec-8-yl.

“Heterocycloalkylene” refers to a heterocyclalkyl group, as definedabove, linking at least two other groups. The two moieties linked to theheterocycloalkylene can be linked to the same atom or different atoms ofthe heterocycloalkylene.

“Aryl” refers to a monocyclic or fused bicyclic, tricyclic or greater,aromatic ring assembly containing 6 to 16 ring carbon atoms. Forexample, aryl may be phenyl, benzyl or naphthyl, preferably phenyl.“Arylene” means a divalent radical derived from an aryl group. Arylgroups can be mono-, di- or tri-substituted by one, two or threeradicals selected from alkyl, alkoxy, aryl, hydroxy, halogen, cyano,amino, amino-alkyl, trifluoromethyl, alkylenedioxy andoxy-C₂-_(C3)-alkylene; all of which are optionally further substituted,for instance as hereinbefore defined; or 1- or 2-naphthyl; or 1- or2-phenanthrenyl. Alkylenedioxy is a divalent substitute attached to twoadjacent carbon atoms of phenyl, e.g. methylenedioxy or ethylenedioxy.Oxy-C₂-_(C3)-alkylene is also a divalent substituent attached to twoadjacent carbon atoms of phenyl, e.g. oxyethylene or oxypropylene. Anexample for oxy-C₂-_(C3)-alkylene-phenyl is 2,3-dihydrobenzofuran-5-yl.

Preferred as aryl is naphthyl, phenyl or phenyl mono- or disubstitutedby alkoxy, phenyl, halogen, alkyl or trifluoromethyl, especially phenylor phenyl-mono- or disubstituted by alkoxy, halogen or trifluoromethyl,and in particular phenyl.

Examples of substituted phenyl groups as R are, e.g. 4-chlorophen-1-yl,3,4-dichlorophen-1-yl, 4-methoxyphen-1-yl, 4-methylphen-1-yl,4-aminomethylphen-1-yl, 4-methoxyethylaminomethylphen-1-yl,4-hydroxyethylaminomethylphen-1-yl,4-hydroxyethyl-(methyl)-aminomethylphen-1-yl 3-aminomethylphen-1-yl,4-N-acetylaminomethylphen-1-yl, 4-aminophen-1-yl, 3-aminophen-1-yl,2-aminophen-1-yl, 4-phenyl-phen-1-yl, 4-(imidazol-1-yl)-phenyl,4-(imidazol-1-ylmethyl)-phen-1-yl, 4-(morpholin-1-yl)-phen-1-yl,4-(morpholin-1-ylmethyl)-phen-1-yl,4-(2-methoxyethylaminomethyl)-phen-1-yl and4-(pyrrolidin-1-ylmethyl)-phen-1-yl, 4-(thiophenyl)-phen-1-yl,4-(3-thiophenyl)-phen-1-yl, 4-(4-methylpiperazin-1-yl)-phen-1-yl, and4-(piperidinyl)-phenyl and 4-(pyridinyl)-phenyl optionally substitutedin the heterocyclic ring.

“Arylene” refers to an aryl group, as defined above, linking at leasttwo other groups. The two moieties linked to the arylene are linked todifferent atoms of the arylene. Arylene groups include, but are notlimited to, phenylene.

“Arylene-oxy” refers to an arylene group, as defined above, where one ofthe moieties linked to the arylene is linked through an oxygen atom.Arylene-oxy groups include, but are not limited to, phenylene-oxy.

Similarly, substituents for the aryl and heteroaryl groups are variedand are selected from: -halogen, —OR′, —OC(0)R\ —NR′R″, —SR′, —R′, —CN,—N0₂, —CO₂R′, —CONR′R″, —C(O)R′, —OC(0)NR′R″, —NR″C(0)R′, —NR″C(0)₂R′,—NR′—C(0)NR″R″′, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH, —NH—C(NH₂)═NR′, —S(0)R′,—S(0)₂R′, —S(0)₂NR′R″, —N₃, —CH(Ph)₂, perfluoro(Ci-C₄)alkoxy, andperfluoro(Ci-C₄)alkyl, in a number ranging from zero to the total numberof open valences on the aromatic ring system; and where R′, R″ and R′″are independently selected from hydrogen, (Ci-Cs)alkyl and heteroalkyl,unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(Ci-C₄)alkyl,and (unsubstituted aryl)oxy-(C₁-C₄)alkyl.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(0)—(CH₂)_(q)—U—, wherein T and U are independently —NH—, -0-, —CH₂—or a single bond, and q is an integer of from 0 to 2. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula-A-(CH₂)_(r)—B—, wherein A and B are independently —CH₂—, -0-, —NH—,—S—, —S(O)—, —S(0)₂—, —S(0)₂NR′— or a single bond, and r is an integerof from 1 to 3. One of the single bonds of the new ring so formed mayoptionally be replaced with a double bond. Alternatively, two of thesubstituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CH₂)_(s)—X—(CH₂)_(t)—, where s and t are independently integers offrom 0 to 3, and X is -0-, —NR′—, —S—, —S(O)—, —S(0)₂—, or —S(0)₂NR′—.The substituent R′ in —NR′— and —S(0)₂NR′— is selected from hydrogen orunsubstituted (Ci-C₆)alkyl.

“Heteroaryl” refers to a monocyclic or fused bicyclic or tricyclicaromatic ring assembly containing 5 to 16 ring atoms, where from 1 to 4of the ring atoms are a heteroatom each N, O or S. For example,heteroaryl includes pyridyl, indolyl, indazolyl, quinoxalinyl,quinolinyl, isoquinolinyl, benzothienyl, benzofuranyl, furanyl,pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl, triazolyl,tetrazolyl, pyrazolyl, imidazolyl, thienyl, or any other radicalssubstituted, especially mono- or di-substituted, by e.g. alkyl, nitro orhalogen. Pyridyl represents 2-, 3- or 4-pyridyl, advantageously 2- or3-pyridyl. Thienyl represents 2- or 3-thienyl. Quinolinyl representspreferably 2-, 3- or 4-quinolinyl. Isoquinolinyl represents preferably1-, 3- or 4-isoquinolinyl. Benzopyranyl, benzothiopyranyl representspreferably 3-benzopyranyl or 3-benzothiopyranyl, respectively. Thiazolylrepresents preferably 2- or 4-thiazolyl, and most preferred,4-thiazolyl. Triazolyl is preferably 1-, 2- or 5-(1,2,4-triazolyl).Tetrazolyl is preferably 5-tetrazolyl.

Preferably, heteroaryl is pyridyl, indolyl, quinolinyl, pyrrolyl,thiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl,thienyl, furanyl, benzothiazolyl, benzofuranyl, isoquinolinyl,benzothienyl, oxazolyl, indazolyl, or any of the radicals substituted,especially mono- or di-substituted.

As used herein, the term “heteroalkyl” refers to an alkyl group havingfrom 1 to 3 heteroatoms such as N, O and S. Additional heteroatoms canalso be useful, including, but not limited to, B, Al, Si and P. Theheteroatoms can also be oxidized, such as, but not limited to, —S(O)—and —S(0)₂—. For example, heteroalkyl can include ethers, thioethers,alkyl-amines and alkyl-thiols.

As used herein, the term “heteroalkylene” refers to a heteroalkyl group,as defined above, linking at least two other groups. The two moietieslinked to the heteroalkylene can be linked to the same atom or differentatoms of the heteroalkylene.

“Electrophile” refers to an ion or atom or collection of atoms, whichmay be ionic, having an electrophilic center, i.e., a center that iselectron seeking, capable of reacting with a nucleophile. Anelectrophile (or electrophilic reagent) is a reagent that forms a bondto its reaction partner (the nucleophile) by accepting both bondingelectrons from that reaction partner.

“Nucleophile” refers to an ion or atom or collection of atoms, which maybe ionic, having a nucleophilic center, i.e., a center that is seekingan electrophilic center or capable of reacting with an electrophile. Anucleophile (or nucleophilic reagent) is a reagent that forms a bond toits reaction partner (the electrophile) by donating both bondingelectrons. A “nucleophilic group” refers to a nucleophile after it hasreacted with a reactive group. Non limiting examples include amino,hydroxyl, alkoxy, haloalkoxy and the like.

“Maleimido” refers to apyrrole-2,5-dione-1-yl group having thestructure:

which upon reaction with a sulfhydryl (e.g., a thio alkyl) forms an—S-maleimido group having the structure

where “.” indicates the point of attachment for the maleimido group and“{circumflex over ( )}” indicates the point of attachment of the sulfuratom the thiol to the remainder of the original sulfhydryl bearinggroup.

For the purpose of this disclosure, “naturally occurring amino acids”found in proteins and polypeptides are L-alanine, L-arginine,L-asparagine, L-aspartic acid, L-cysteine, L-glutamine, L-glutamic acid,L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine,L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan,L-tyrosine, and or L-valine. “Non-naturally occurring amino acids” foundin proteins are any amino acid other than those recited as naturallyoccurring amino acids. Non-naturally occurring amino acids include,without limitation, the D isomers of the naturally occurring aminoacids, and mixtures of D and L isomers of the naturally occurring aminoacids. Other amino acids, such as 4-hydroxy proline, desmosine,isodesmosine, 5-hydroxy lysine, epsilon-N-methyllysine,3-methylhistidine, although found in naturally occurring proteins, areconsidered to be non-naturally occurring amino acids found in proteinsfor the purpose of this disclosure as they are generally introduced bymeans other than ribosomal translation of mRNA.

“Linear” in reference to the geometry, architecture or overall structureof a polymer, refers to polymer having a single polymer arm.

“Branched,” in reference to the geometry, architecture or overallstructure of a polymer, refers to a polymer having 2 or more polymer“arms” extending from a core structure contained within an initiator.The initiator may be employed in an atom transfer radical polymerization(ATRP) reaction. A branched polymer may possess 2 polymer chains (arms),3 polymer arms, 4 polymer arms, 5 polymer arms, 6 polymer arms, 7polymer arms, 8 polymer arms, 9 polymer arms or more. Each polymer armextends from a polymer initiation site. Each polymer initiation site iscapable of being a site for the growth of a polymer chain by theaddition of monomers. For example and not by way of limitation, usingATRP, the site of polymer initiation on an initiator is typically anorganic halide undergoing a reversible redox process catalyzed by atransition metal compound such as cuprous halide. Preferably, the halideis a bromine.

“Pharmaceutically acceptable” composition or “pharmaceuticalcomposition” refers to a composition comprising a compound of theinvention and a pharmaceutically acceptable excipient orpharmaceutically acceptable excipients.

“Pharmaceutically acceptable excipient” and “pharmaceutically acceptablecarrier” refer to an excipient that can be included in the compositionsof the invention and that causes no significant adverse toxicologicaleffect on the patient and is approved or approvable by the FDA fortherapeutic use, particularly in humans. Non-limiting examples ofpharmaceutically acceptable excipients include water, NaCl, normalsaline solutions, lactated Ringer's, normal sucrose, normal glucose andthe like.

“Patient” or “subject in need thereof refers to a living organismsuffering from or prone to a condition that can be prevented or treatedby administration of a pharmaceutical composition as provided herein.Non-limiting examples include humans, other mammals and othernon-mammalian animals.

Conjugates are preferably provided in isolated form. Isolated means thatan object species has been at least partially separated fromcontaminants with which it is naturally associated or which are used inits manufacture but does not necessarily exclude the presence of othercomponents intended to act in combination with an isolated species, suchas a pharmaceutical excipient. Preferably a conjugate is the predominantmacromolecular species present in a sample (i.e., on a molar basis in acomposition and typically comprises at least about 50 percent (cm amolar basis) of all macromolecular species present. Generally, anisolated conjugate comprises more than 80, 90, 95 or 99 percent of allmacromolecular species present in a composition. Most preferably, aconjugate is purified to essential homogeneity (i.e., contaminantspecies cannot be detected in a composition by conventional detectionmethods), such that the composition consists essentially of a singlemacromolecular species. Conjugates have the same heavy and light chainsare considered to be the same species notwithstanding there may bevariation in glycosylation on protein moieties and variation in numbersof monomers in polymer moieties linked to different molecules of theconjugate.

“Therapeutically effective amount” refers to an amount of a conjugatedfunctional agent or of a pharmaceutical composition useful for treating,ameliorating, or preventing an identified disease or condition, or forexhibiting a detectable therapeutic or inhibitory effect. The effect canbe detected in an individual patient relative to a baseline measurementbefore treatment or by determining a statistically significantdifference in outcome between treated and control populations.

The “biological half-life” of a substance is a pharmacokinetic parameterwhich specifies the time required for one half of the substance to beremoved from an organism following introduction of the substance intothe organism.

Sequence identity can be determined by aligning sequences usingalgorithms, such as BESTFIT, PASTA, and TFASTA in the Wisconsin GeneticsSoftware Package Release 7.0, Genetics Computer Group, 575 Science Dr.,Madison, Wis., using default gap parameters, or by inspection, and thebest alignment (i.e., resulting in the highest percentage of sequencesimilarity over a comparison window). Percentage of sequence identity iscalculated by comparing two optimally aligned sequences over a window ofcomparison, determining the number of positions at which the identicalresidues occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the window of comparison (i.e., the window size), andmultiplying the result by 100 to yield the percentage of sequenceidentity.

III. Factor VIII

Coagulation factor VIII (FVIII) circulates in plasma at a very lowconcentration and is bound non-covalently to von Willebrand factor(VWF). During hemostasis, FVIII is separated from VWF and acts as acofactor for activated factor FX (FFXa)-mediated factor X (FX)activation by enhancing the rate of activation in the presence ofcalcium and phospholipids or cellular membranes.

FVIII is synthesized as a single-chain precursor of approximately270-330 kDa with the domain structure A1-A2-B-A3-C1-C2 (FIG. 2 ). Whenpurified from plasma (e.g., “plasma-derived” or “plasmatic”), FVIII iscomposed of a heavy chain (A1-A2-B) and a tight chain (A3-C1-C2). Themolecular mass of the light chain is 80 kDa whereas, due to proteolysiswithin the B domain, the heavy chain is in the range of 90-220 kDa. Thedomains are delineated as follows in SEQ ID NO:1 A1, residues Ala 1-Arg372; A2, residues Ser 373-Arg 740; B, residues Ser 741-Arg 1648; A3,residues Ser 1690-Ile 2032; CI, residues Arg 2033-Asn 2172; and C2,residues Ser 2173-Tyr 2332. The remaining sequence, residues Glu1649-Arg 1689, is usually referred to as the factor VIII light chainactivation peptide. Factor VIII is proteolytically activated by thrombinor factor Xa, which dissociates it from von Willebrand's factor, formingfactor Villa, which has procoagulant function. The biological functionof factor Villa is to increase the catalytic efficiency of factor IXatoward factor X activation by several orders of magnitude.Thrombin-activated factor Villa is a 160 kDa A1/A2/A3-C1-C2.

Mature Factor VIII is heavily glycosylated and proteolyzed andcirculates as a hetero-dimer having a heavy chain and a light chainconnected by metal ions. The heavy chains consists of sequence relatedregions A1 and A2 domains and a connecting B region, which is heavilyglycosylated. The light chain consists of A3, C1 and C2 domains. Inplasma, Factor VIII circulates as a non-covalent complex with VonWillebrand's factor. It has been found that the B domain is unnecessaryfor Factor VIII coagulation activity.

Various in vitro assays have been devised to determine the potentialefficacy of recombinant FVIII (rFVIII) as a therapeutic medicine. Theseassays mimic the in vivo effects of endogenous FVIII. In vitro thrombintreatment of FVIII results in a rapid increase and subsequent decreasein its procoagulant activity, as measured by in vitro assay. Thisactivation and inactivation coincides with specific limited proteolysisboth in the heavy and the light chains, which alter the availability ofdifferent binding epitopes in FVIII, e.g. allowing FVIII to dissociatefrom VWF and bind to a phospholipid surface or altering the bindingability to certain monoclonal antibodies.

The production of recombinant Factor VIII by recombinant engineeringtechniques has been described. See, e.g., U.S. Pat. Nos. 4,757,006,5,733,873, 5,198,349, 5,250,421, 5,919,766 and European Patent No. 306968. The gene for Factor VIII is located on the tip of the long arm ofthe X chromosome. The human Factor VIII gene comprises 26 exons spreadout over 186,000 bp of genomic DNA and codes for a protein of 2351 aminoacids, including a 19 amino acid leader sequence. Factor VIII is one ofthe largest known genes. The mature Factor VIII protein is 2332 aminoacids (Swiss Prot P00451). The protein sequence of Factor VIII is setforth in FIG. 1 . FVIII is considered to be recombinant if synthesizedas a result of genetic techniques as distinct from being isolated from anatural source, such as human plasma. Recombinant FVIII may or may notbe changed in other respects from plasma derived FVIII (e.g., bytruncation, or mutation).

FVIII is subject to numerous known polymorphisms described in the SwissProt database. Thus, for example, the aspartic acid residue at position56 may optionally be valine in accordance with the present invention.Similarly, the aspartic acid at position 1141 may also be glutamic acidin accordance with the present invention. All known or discoveredallelic and polymorphic variations of FVIII are included within thescope of the present invention.

Herein, the term “Factor VIII” or “FVIII” refers to any FVIII moleculewhich exhibits biological activity, particularly promotion of bloodclotting, that is associated with native FVIII. Several assays for FVIIIactivity are commercially available (see Chandler et al., Am J ClinPathol 2003; 120:34-39). In a preferred embodiment of the presentinvention, the FVIII has al least a portion or all of the B domain(e.g., at least 100, 200, 500 or 900 residues including at least onecysteine conjugatable to a polymer). Preferably the portion includes thetwo most C-terminal cysteines from the intact B-domain (at positions1604 and 1636). In one embodiment of the invention, the FVIII moleculeis full-length Factor VIII (except the signal peptide can be deleted).The FVIII molecule is a protein which is encoded for by DNA sequencescapable of hybridizing to DNA encoding Factor VIII:C under stringentconditions (e.g., 52° C. 50% formamide, 5×SSC). Such a protein maycontain amino acid deletions at various sites between or within thedomains A1-A2-B-A3-C1-C2 (see, e.g., U.S. Pat. No. 4,868,112). The FVIIImolecule may also be an analog of native FVIII wherein one or more aminoacid residues have been replaced by site-directed mutagenesis.

The FVIII molecules useful for the present invention include thefull-length protein, precursors of the protein, biologically active orfunctional subunits or fragments of the protein, and functionalderivatives thereof, as well as variants thereof as described hereinbelow. Reference to FVIII is meant to include all potential forms ofsuch proteins and including forms of FVIII having at least a portion orall of the native B domain sequence intact and forms in which the Bdomain is absent.

In another aspect of the present invention, FVIII moieties havingvarious deletions may also be conjugated to the polymers of the presentinvention. The Factor VIII molecules according to this aspect of thepresent invention are B domain truncated Factor FVIII wherein theremaining domains have the sequence as set forth in amino acid no 1-740and 1649-2332 in SEQ ID NO. 1. Factor VIII molecules according to theinvention are preferably recombinant molecules produced in transformedhost cells, preferably of mammalian origin.

However, the remaining domains (i.e. the three A-domains and the twoC-domains) may differ slightly e.g. about 1%, 2%, 0.3%, 4% or 5% fromthe amino acid sequence as set forth in SEQ ID NO 1 (amino acids 1-740and 1649-2332). In particular, amino acid modifications (substitutions,deletions, or insertions) can be introduced in the remaining domains,e.g. in order to modify the binding capacity of Factor VIII with variousother components such as e.g. vW factor, LPR, various receptors, othercoagulation factors, cell surfaces, etc. Furthermore, the Factor VIIImolecules according to the invention can comprise otherpost-translational modifications in e.g. the truncated B-domain and/orin one or more of the other domains of the molecules. These otherpost-translational modifications may be in the form of various moleculesconjugated to the Factor VIII molecule according to the invention suchas e.g. polymeric compounds, peptidic compounds, fatty acid derivedcompounds, and so forth.

Factor VIII molecules according to the present invention, regardless ofwhether they are modified outside the B domain or not, have otherpost-translational modifications or not, all have Factor VIII activity,meaning the ability to function in the coagulation cascade in a mannerfunctionally similar or equivalent to FVIII, induce the formation of FXavia interaction with FIXa on an activated platelet and support theformation of a blood clot. The activity can be assessed in vitro bytechniques well known in the art (e.g., Chandler et al., supra) such ase.g. clot analysis, endogenous thrombin potential analysis, etc. FactorVIII molecules according to the present invention have FVIII activitybeing at least about 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, and100% or even more than 100% of that of native human FVIII.

The B-domain in Factor VIII spans amino acids 741-1648 in SEQ ID NO 1.The B-domain is cleaved at several different sites, generating largeheterogeneity in circulating plasma FVIII molecules. The exact functionof the heavily glycosylated B-domain is unknown. But the domain isdispensable for FVIII activity in the coagulation cascade. This apparentlack of function is supported by the fact that B domaindeleted/truncated FVIII appears to have in vivo properties identical tothose seen for full length native FVIII. That being said there areindications that the B-domain may reduce the association with the cellmembrane, at least under serum free conditions.

B domain truncated/deleted Factor VIII molecule: Endogenous full lengthFVIII is synthesized as a single-chain precursor molecule. Prior tosecretion, the precursor is cleaved into the heavy chain and the lightchain. Recombinant B domain-deleted FVIII can be produced from twodifferent strategies. Either the heavy chain without the B-domain andthe light chain are synthesized individually as two differentpolypeptide chains (two-chain strategy) or the B-domain deleted FVIII issynthesized as a single precursor polypeptide chain (single-chainstrategy) that is cleaved into the heavy and light chains in the sameway as the full-length FVIII precursor.

In a B domain-deleted FVIII precursor polypeptide, the heavy and lightchain moieties are normally separated by a linker. To minimize the riskof introducing immunogenic epitopes in the B domain-deleted FVIII, thesequence of the linker is preferably derived from the FVIII B-domain.The linker must comprise a recognition site for the protease thatseparates the B domain-deleted FVIII precursor polypeptide into theheavy and light chain. In the B domain of full length FVIII, amino acids1644-1648 constitute this recognition site. The thrombin site leading toremoval of the linker on activation of B domain-deleted FVIII is locatedin the heavy chain. Thus, the size and amino acid sequence of the linkeris unlikely to influence its removal from the remaining FVIII moleculeby thrombin activation. Deletion of the B domain is an advantage forproduction of FVIII. Nevertheless, parts of the B domain can be includedin the linker without reducing the productivity. The negative effect ofthe B domain on productivity has not been attributed to any specificsize or sequence of the B domain.

According to the present invention, the term “recombinant Factor VIII”(rFVIII) may include any rFVIII, heterologous or naturally occurring,obtained via recombinant DNA technology, or a biologically activederivative thereof. In certain embodiments, the term encompassesproteins as described above and nucleic acids, encoding a rFVIII of theinvention. Such nucleic acids include, for example and withoutlimitation, genes, pre-mRNAs, mRNAs, polymorphic variants, alleles,synthetic and naturally-occurring mutants. Proteins embraced by the termrFVIII include, for example and without limitation, those proteins andpolypeptides described hereinabove, proteins encoded by a nucleic aciddescribed above, interspecies homologs and other polypeptides that havean amino acid sequence that has greater than about 85%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98% or about 99% or greater amino acid sequence identity, over aregion of at least about 100, about 200, about 300, about 400, or moreamino, to a polypeptide encoded by a referenced nucleic acid or an aminoacid sequence described herein. Preferably. Factor VIII shows at least90, 95, 96, 97, 98 or 99% sequence identity to the entire sequence ofA1, A2, A3, C1, and C2 domains.

In accordance with certain aspects of the present invention, productionof rFVIII includes any method known in the art for (i) the production ofrecombinant DNA by genetic engineering, (ii) introducing recombinant DNAinto prokaryotic or eukaryotic cells by, for example and withoutlimitation, transfection, electroporation or microinjection, (iii)cultivating said transformed cells, (iv) expressing rFVIII, e.g.constitutively or upon induction, and (v) isolating said rFVIII, e.g.from the culture medium or by harvesting the transformed cells, in orderto (vi) obtain purified rFVIII.

In preferred aspects of the present invention, the rFVIII is produced byexpression in a suitable prokaryotic or eukaryotic host systemcharacterized by producing a pharmacologically acceptable rFVIIImolecule. Examples of eukaryotic cells are mammalian cells, such as CHO,COS, HEK 293, BHK, SK-Hip, and HepG2.

In still other aspects, a wide variety of vectors are used for thepreparation of the rFVIII and are selected from eukaryotic andprokaryotic expression vectors. Examples of vectors for prokaryoticexpression include plasmids such as, and without limitation, preset,pet, and pad, wherein the promoters used in prokaryotic expressionvectors include one or more of, and without limitation, lac, trc, tip,recA, or araBAD. Examples of vectors for eukaryotic expression include:(i) for expression in yeast, vectors such as, and without limitation,pAO, pPIC, pYES, or pMET, using promoters such as, and withoutlimitation, AOX1, GAP, GAL1, or AUG1; (ii) for expression in insectcells, vectors such as and without limitation, pMT, pAc5, pIB, pMIB, orpBAC, using promoters such as and without limitation PH, plO, MT, Ac5,OpIE2, gp64, or polh, and (iii) for expression in mammalian cells,vectors such as, and without limitation, pSVL, pCMV, pRc/RSV, pcDNA3, orpBPV, and vectors derived from, in one aspect, viral systems such as andwithout limitation vaccinia virus, adeno-associated viruses, herpesviruses, or retroviruses, using promoters such as and without limitationCMV, SV40, EF-1, UbC, RSV, ADV. BPV, and beta-actin.

FVIII molecules may be coupled to polymers of the instant invention asdescribed herein for other functional agents, including proteins. Forexample, in one embodiment polymer is conjugated to FVIII via free aminogroups of the protein using N-hydroxysuccinimide (NHS) esters. Reagentstargeting conjugation to amine groups can randomly react to e-aminegroup of lysines, a-amine group of N-terminal amino acids, and 8-aminegroup of histidines. Full length FVIII has 158 lysines, 2N-termini, and75 histidines. In accordance with an aspect of the present invention,conjugates can be formed using one or more of these sites. However, itis known that FVIII is required to interact with multiple partners suchas von Willebrand Factor (VWF), coagulation factor X (FX), and activatedfactor EX (FEXa) for full activity. Conjugation of polymers to freeamino groups, thus, might negatively impact the ability of theconjugated FVIII to affect clotting.

In another embodiment the polymers of the instant invention may becoupled to free SH groups using any appropriate thiol-reactive chemistryincluding, without limitation, maleimide chemistry, or the coupling ofpolymer hydrazides or polymer amines to carbohydrate moieties of theFVIII after prior oxidation. The use of maleimide coupling is aparticularly preferred embodiment of the present invention.

In accordance with a preferred embodiment of the present invention,polymers may be coupled to any cysteine residue of FVIII using maleimidecoupling as long as sufficient biological activity is retained.Alternatively, any suitable amine or carbohydrate moiety of FVIII may beused for coupling of the polymers of the instant invention to FVIII aslong as sufficient activity is retained.

FVIII has 4 cysteines in the B domain and 19 cysteines in the otherdomains. Of the 19 cysteines in B domain-deleted (BDD) FVIII, 16 formdisulfides and the other 3 are free cysteines. The structural model ofBDD-FVIII suggests that all 3 free cysteines are buried and would not beaccessible for reaction with a polymer (Baisan et al. 116 Blood 270-279(2010)). Thus, in accordance with an aspect of the present invention,polymers are preferably covalently attached to cysteine residuesintroduced into FVIII by site directed mutagenesis (see Table 1, belowfor possible sites). See, e.g., EP 2363414A2.

TABLE 1 FVIII Cysteine Variants for Zwitterionic Polymer Conjugation CysMutation Domain Y81C A1 F129C A1 K377C A2 H378C A2 K422C A2 Q468C A2L491C A2 L504C A2 K556C A2 K570C A2 D1795C A3 Q1796C A3 R1803C A3 K1804CA3 K1808C A3 K1810C A3 T1812C A3 K1813C A3 N1864C A3 T1911C A3 N2118C CIQ2091C CI Q2284C C2See, e.g. Mei, B., et al., (2012) Thrombosis and Hemostasis 116,270-279.

According to another aspect of the present invention, polymers arepreferably coupled to cysteine residues naturally occurring in the Bdomain. Alternatively, in accordance with an aspect of the presentinvention, cysteine residues may be added to the B domain viarecombinant DNA technology. The polymer can be conjugated to the rFVIIIvia one and only one cysteine residue, or via multiple cysteines.Preferably the cysteine(s) are in the B domain, preferably either of thetwo most C-terminal cysteines in the B-domain at positions 1604 and 1636of SEQ ID NO:1. (If only a portion of the B-domain is used, the two mostC-terminal cysteines are the C-terminal cysteines in the portion alignedwith the two most C-terminal cysteines of the intact B-domain.)Attachment of a branched polymer via a single cysteine in any givenmolecule of rFVIII is advantageous for surrounding rFVIII with polymerand zwitterionic charge without substantial impairment if any of rFVIIIIactivity. The single cysteine via which the polymer is conjugated can bethe same or different in different molecules of rFVIIII. Although anunderstanding of mechanism is not required for practice of theinvention, it is believed that the zwitterionic charge on polymersurrounding rFVIII virtually immobilizes a layer of water molecules thatconsequently moves in tandem with rFVIII protecting it from degradativeprocesses in vivo.

A preparation of rFVIII is typically homogenous due to proteoloyticprocessing at different sites within the B domain resulting in severalbands from the heavy chain on a gel. Conjugation of such a preparationof rFVIII to a polymer of the invention resulting in polymerization viaone of the two C-terminal cysteines forms conjugates only for moleculesof rFVIII in which these two-terminal cysteines are present. Moleculesof rFVIII in which the B domain is more truncated do not form conjugatesto a significant extent. Specificity of conjugation to a single form ofB-domain is shown by comparing the bands on a gel before and afterconjugation and observing loss or substantial reduction of only one ofthe bands pre-conjugation. Conjugated rFVIII can easily be separatedfrom unconjugated molecules of rFVIII due to the large difference inmolecular weight. In consequence, a preparation of conjugated rFVIII canhave much greater homogeneity than a typical preparation of rFVIII. Forexample, at least 80, 90, 95 or 99% of molecules in a preparation canhave the same portion of the B-domain, for example, an intact B-domainand one and only one polymer (preferably branched) attached per molecule(although there may be glycosylation differences between differentproteins and difference in length between different polymers). In somepreparations, the portion is at least residues 1-1604 or 1-1636 or1-1648 of SEQ ID NO:1. In some preparations, the portions consists ofresidues 1-1648 of SEQ ID NO:1.

The B-domain of a conjugate including the polymer linked to it may beexcised after administration to a subject by the endogenous FVIIIactivation process. However, the conjugated polymer still fulfills arole of prolonging the half-life of the linked FVIII until a need foractivity occurs. Moreover, loss of polymer in the course of activationhas the advantage that FVIII can be more rapidly degraded (compared withFVIII conjugated other than via the B domain) after activation hasoccurred. For this reason, rFVIII conjugated via the B domain isadvantageous for prophylaxis to subjects having hemophilia but not knownto be experiencing bleeding (internal or external) at the time ofadministration. Conjugation to the polymer facilitates persistence ofthe conjugate until such time as the subject may be determined to beexperiencing a bleeding episode.

At this time, the B-domain and associated polymer may be processed fromthe rFVIII, and the remaining rFVIII can facilitate clotting andthereafter be inactivated.

Conjugation of rFVIII to a polymer according to the present methodsincreases the in vivo half-life of rFVIII in humans above 11 hours. Forexample, the half-life can be 12-50 hours. Preferably, the half-life is20 hours or longer. Half-lives are measured as means in a population ofhuman subjects free of prior antibody response to human FVIII. Suchhuman can have but need not have hemophilia for purposes of determininghalf-life.

Because of its longer half-life, the conjugate can be administered lessfrequently in prophylaxis than in current regimes, for example,administration at no more than weekly intervals. In some prophylacticregimes, the conjugate is administered at a frequency between weekly andmonthly, for example, weekly, biweekly, or monthly. Despite thedecreased frequency of administration, subjects receiving the conjugatecan have increased trough levels of FVIII compared with current methods.In current methods, subjects on prophylactic regimes spend about 18 hourper week with trough levels of FVIII activity at a level below 1% ofthat of the mean level in control subjects without hemophilia. A levelbelow 1%, places a subject at high risk of an acute bleed. With thepresent methods, subjects can be maintained with a trough level above1%, 3% or even 5% of the mean level of FVIII activity in controlsubjects without hemophilia for a period of at least a week, a month, ayear or indefinitely. Activity can be assessed in an in vitrochromogenic assay, which includes activation of FVIII by processing ofthe B domain.

In prophylactic treatment or other treatment, the conjugate of theinvention is suitable for administration to subjects who have previouslybeen treated with FVIII (not conjugated as described herein) anddeveloped a human antibody response against it. The polymer moiety ofthe present conjugates shields the FVIII of the present conjugates fromsuch antibodies allowing it to persist in the blood for longer thanwould be the case for unconjugated FVIII, and preferably withessentially the same half-life in a subject without antibodies to FVIII.

With regard to the naturally occurring cysteines in the B domain, anintact B domain is not essential for FVIII activity. The B domain ofFVIII begins at amino acid 745 and continues to amino acid 1648. The Bdomain has 4 naturally occurring cysteine residues: 1293, 1373, 1604 and1636. In accordance with the present invention, coupling at one or moreof these residues is preferred. Coupling of the polymers of the presentinvention to residues 1604 and 1636 is particularly preferred.

In accordance with the present invention, conjugates of the high MWpolymers of the present invention and FVIII are presented. In accordancewith one aspect of the present invention, preferred conjugates arepresented in which FVIII is coupled to a zwitterionic polymer whereinthe polymer is composed of one or more monomer units and wherein atleast one monomer unit has a zwitterionic group. Preferably, thezwitterionic group is phosphorylcholine.

In a preferred aspect of the present invention, one of the monomer unitsis 2-(acryloyloxyethyl)-2′-(trimethylammoniumethyl) phosphate or2-(methacryloyloxyethyl)-2′-(trimethylammoniumethyl) phosphate(HEMA-PC). In other preferred embodiments, polymer is synthesized from asingle monomer which is preferably2-(acryloyloxyethyl)-2′-(trimethylammoniumethyl) phosphate or2-(methacryloyloxyethyl)-2′-(trimethylammoniumethyl) phosphate.

In a preferred embodiment of the present invention, the FVIII or theconjugate is a recombinant FVIII (rFVIII). In preferred embodiments ofthe present invention, rFVIII is full length. In other preferredembodiments of the present invention, the rFVIII is purified from amammalian host cell. In still another aspect of the present invention,the FVIII comprises a deletion of part or all of the B domain.

In still other aspects of the present invention, it is preferred thatthe FVIII conjugates have 2 or more preferably 3 or more polymer armswherein the monomer is HEMA-PC. In another aspect of the presentinvention, it is preferred that the conjugates have 2, 3, 4, 5, 6, 7, 8,9, 10, 11, or 12 polymer arms wherein the monomer is HEMA-PC. Morepreferably, the conjugates have 3, 6 or 9 arms. Most preferably, theconjugate has 9 arms.

In one aspect of the present invention, it is preferred that thepolymer-FVIII conjugates have a polymer portion with a molecular weightof between 100,000 and 1,500,000 daltons. More preferably the conjugatehas a polymer portion with a molecular weight between 500,000 and1,000,000 daltons. Still more preferably the conjugate has a polymerportion with a molecular weight between 600,000 to 800,000 daltons. Mostpreferably the FVIII conjugate has a polymer portion with a molecularweight between 600.000 and 850,000 daltons and has 9 arms. Here andelsewhere in this application, the total molecular weight for polymerincluding the FVIII is in ranges about 300,000 daltons higher than thosegiven for the polymer portion.

In accordance with an aspect of the present invention, methods areprovided for synthesizing a zwitterionic polymer-functional agentconjugate, the conjugate having one or more functional agents and one ormore polymer arms wherein each of the polymer arms has one or moremonomer types wherein at least one of the types has a zwitterion.According to an aspect of the present invention, the method has thesteps of

-   -   a. combining an initiator comprising one or more polymer        synthesis initiator moieties and a first reactive group with one        or more monomer types suitable for polymerization wherein at        least one of said monomer types comprises a zwitterion; wherein        said monomer types react to form polymer(s) at the polymer        synthesis initiator moity(ies) to provide a polymerized        initiator;    -   b. coupling a linker moiety comprising second and third reactive        groups to the polymerized initiator to provide a        linker-polymerized initiator having an unreacted reactive group;        and    -   c. coupling one or more functional agents to the unreacted        reactive group of the linker-polymerized initiator to provide        the polymer-functional agent conjugate.

Prior to the instant invention, the initiator molecule or entity had tocontain a deprotectable functional group that would allow coupling ofthe functional agent. An example of such an initiator having a protectedmaleimide is shown below:

After polymer synthesis, the protected maleimide is deprotected withheat to allow for generation of maleimide which could be used to couplefunctional agent. If one wanted to vary the nature of the chemicalentity in between the maleimide and the polymer initiation site, onewould have to synthesize an entire new initiator.

Considering possible scale up of the polymer synthesis process, eachtime the initiator is changed or altered in any way, a new scaled upsynthesis procedure has to be developed. Each change in the nature ofthe initiator molecule can have a wide range of effects on polymersynthesis. However, in accordance with the present invention, a singleinitiator moiety can be used for large scale polymer preparation. Thus,conditions can be developed for scaled up optimal polymer synthesis.Using the instantly claimed invention, such polymer can then be adaptedto various types of functional agents by “snapping-on” various types oflinkers.

For example, if it is desired to conjugate a larger functional agent toa polymer of the instant invention such as an antibody of even a Fabfragment, a longer linker sequence can be snapped on to the poly mer. Incontrast, smaller functional agents may call for relatively shorterlinker sequences.

In preferred embodiments of the methods, the initiator has 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11 or 12 sites for polymer initiation. Preferably,the initiator has 3, 6 or 9 sites for polymer initiation.

In accordance with this aspect of the present invention, poly mersynthesis imitator moieties preferably have the following structure:

where X is a NCS or a halogen which allows initiation of ATRP or relatedpolymer synthesis schemes and R is the rest of the imitator.

In accordance with the present invention, the initiator preferably hasthe structure:R₁—R₂

R₃)_(s)wherein R1 has a nucleophilic reactive group, R2 comprises a linker, andR3 is a polymer synthesis initiator moiety and s is an integer between 1and 20. R1 is preferably selected from the group consisting of NH₂—,OH—, and SH. More preferably, R1 is NH₂—.

In accordance with this aspect of the present invention, R2 ispreferably alkyl, substituted alkyl, alkylene, alkoxy, carboxyalkyl,haloalkyl, cycloalkyl, cyclic alkyl ether, alkenyl, alkenylene, alkynyl,alkynylene, cycloalkylene, heterocycloalkyl, heterocycloalkylene, arylarylene, arylene-oxy, heteroaryl, amino, amido or any combinationthereof.

More preferably, R2 comprises a structure having the formula:

wherein m is 1 to 20, m is preferably 4.

In accordance with the present invention, R3 preferably has thefollowing formula

wherein R4, R5 and R6 are the same or different and are selected fromthe group consisting of

wherein X is NCS, F, CI, Br or I. Preferably X is Br.

In more preferred aspects of the present invention, R4, R5 and R6 areeach

Alternatively, R4, R5 and R6 are each

In still other preferred embodiments, R4, R5 and R6 are each

In accordance with this aspect of the present invention, the monomer ispreferably selected from the group consisting of

wherein R7 is H or Ci_6 alkyl, ZW is a zwitterion and t is 1 to 6.Preferably, the zwitterion is phosphorylcholine.

Still more preferably, the monomer is selected from the group consistingof 2-(methacryloyloxyethyl)-2′-(trimethylammoniumethyl) phosphate(HEMA-PC) and 2-(acryloyloxyethyl)-2′-(trimethylammoniumethyl)phosphate. Most preferably, the monomer is2-(methacryloyloxyethyl)-2′-(trimethylammoniumethyl) phosphate.

In accordance with an aspect of the present invention, the linker moietyof step d is preferably an activated ester having the structure

wherein R8 is selected from the group consisting of

and R9 is selected from the group consisting of

wherein p is 1 to 2.

Preferably, the linker moiety is

In accordance with an aspect of the present invention, the initiator ofstep a. preferably has the following structure:

wherein y is an integer from 1 to 50, X is an integer from 0 to 50 and Zis NCS, F, Cl, Br or I. Preferably, Z is Br, X is 4, 8 or 12 and Y is 1to 10. More preferably, Y is 4.

In accordance with this aspect of the present invention, thelinker-polymerized initiator of step f preferably has the formula:

wherein X is an integer from 1 to 50, Y is an integer from 1-50 andPolymer is any polymer synthesized with a monomer as defined herein.More preferably Y is 4, X is 4, 8 or 12 and the monomer is HEMA-pc.

Preferably, the functional agent is a protein. More preferably, theprotein comprises human FVIII. Still more preferably, the FVIII is arecombinant FVIII (rFVIII) which is preferably purified from a humanhost cell. Most preferably, the FVIII has a deletion of part or all ofthe B domain.

In accordance with an aspect of the present invention, a compound ispresented having the formula:

wherein y is an integer from 1 to 50, X is an integer from 0 to 50 and Zis NCS, F, Cl, Br or I. Preferably, Z is Br, X is 4, 8 or 12 and Y is 1to 10. More preferably, Y is 4.

In accordance with another aspect of the present invention, a polymer ispresented having the formula:

wherein y is an integer from 1 to 50. X is an integer from 0 to 50 andMPC is a poly MPC arm. Poly MPC is prepared using is2-(methacryloyloxyethyl)-2′-(trimethylammoniumethyl) phosphate in apolymerization reaction, e.g., ATRP. Preferably, the total molecularweight of the polymer is about 500,000 to about 1,000,000 Daltons. Morepreferably, the total molecular weight of the polymer is about 650,000to about 850,000 daltons. Still more preferably, the total molecularweight of the polymer is about 750,000 daltons.

In accordance with this aspect of the present invention X is preferably4, 8 or 12 and Y is 1 to 10. Still more preferably, Y is 4.

Such an initiator be used, in accordance with the present invention asthe substrate for polymer synthesis. Preferably, the polymer synthesisis conducted using ATRP or like method, such as generated by AGET(Woodworth et al., Macromolecules, Vol. 31, No. 23, 1998) or ARGET(Macromolecules, 2012, 45 (16), pp 6371-6379 (Simakova, A. et al)). Anyof the monomers described herein may be used for polymer synthesis.

Pharmaceutical compositions adapted for oral administration may bepresented as discrete units such as capsules, as solutions, syrups orsuspensions (in aqueous or non-aqueous liquids; or as edible foams orwhips; or as emulsions). Suitable excipients for tablets or hardgelatine capsules include lactose, maize starch or derivatives thereof,stearic acid or salts thereof. Suitable excipients for use with softgelatine capsules include for example vegetable oils, waxes, fats,semi-solid, or liquid polyols etc. For the preparation of solutions andsyrups, excipients which may be used include for example water, polyolsand sugars. For the preparation of suspensions oils (e.g. vegetableoils) may be used to provide oil-in-water or water in oil suspensions.

Pharmaceutical compositions adapted for nasal administration wherein thecarrier is a solid include a coarse powder having a particle size forexample in the range 20 to 500 microns which is administered in themanner in which snuff is taken, i.e. by rapid inhalation through thenasal passage from a container of the powder held close up to the nose.Suitable compositions wherein the carrier is a liquid, foradministration as a nasal spray or as nasal drops, include aqueous oroil solutions of the active ingredient. Pharmaceutical compositionsadapted for administration by inhalation include fine particle dusts ormists which may be generated by means of various types of metered dosepressurized aerosols, nebulizers or insufflators.

Pharmaceutical compositions adapted for parenteral administrationinclude aqueous and non-aqueous sterile injection solution which maycontain anti-oxidants, buffers, bacteriostats and solutes which renderthe formulation substantially isotonic with the blood of the intendedrecipient; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents and thickening agents. Excipients which may beused for injectable solutions include water, alcohols, polyols,glycerine and vegetable oils, for example. The compositions may bepresented in unit-dose or multi-dose containers, for example scaledampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carried, forexample water for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions may be prepared from sterilepowders, granules and tablets. Pharmaceutical compositions can besubstantially isotonic, implying an osmolality of about 250-350 mOsm/kgwater.

In general, the pharmaceutical compositions may contain preservingagents, solubilizing agents, stabilizing agents, wetting agents,emulsifiers, sweeteners, colorants, odorants, salts (substances of thepresent invention may themselves be provided in the form of apharmaceutically acceptable salt), buffers, coating agents orantioxidants. They may also contain therapeutically active agents inaddition to the substance of the present invention. The pharmaceuticalcompositions of the invention may be employed in combination withpharmaceutically acceptable diluents, adjuvants, or carriers. Suchexcipients may include, but are not limited to, saline, buffered saline(such as phosphate buffered saline), dextrose, liposomes, water,glycerol, ethanol and combinations thereof.

The pharmaceutical compositions may be administered in any effective,convenient manner effective for treating a patients disease including,for instance, administration by oral, intravenous, subcutaneous,intramuscular, intraosseous, intranasal, or routes among others. Intherapy or as a prophylactic, the active agent may be administered to anindividual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage of the active agent will be from 0.01 mg/kg bodyweight typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual which will be dependent on factors including the age, weight,sex and response of the individual. The above dosages are exemplary ofthe average case. There can, of course, be instances where higher orlower dosages are merited, and such are within the scope of thisinvention.

Dosages of the substance of the present invention can vary between widelimits, depending upon the disease or disorder to be treated, the ageand condition of the individual to be treated, etc. and a physician willultimately determine appropriate dosages to be used.

This dosage may be repeated as often as appropriate. If side effectsdevelop the amount and/or frequency of the dosage can be reduced, inaccordance with normal clinical practice. In one embodiment, thepharmaceutical composition may be administered once every one to thirtydays.

According to a third aspect of the invention, there is provided apharmaceutical composition of the second aspect and anotherpharmaceutically active agent. The other pharmaceutically active agentmay promote or enhance the activity of FVIII, for example another bloodcoagulation factor.

The pharmaceutical compositions of the invention may be employed aloneor in conjunction with other compounds, such as therapeutic compounds ormolecules, e.g. anti-inflammatory drugs, analgesics or antibiotics. Suchadministration with other compounds may be simultaneous, separate orsequential. The components may be prepared in the form of a kit whichmay comprise instructions as appropriate.

Preferably, the pharmaceutical composition of the invention and theother therapeutic compound are directly administered to a patient inneed thereof.

The invention also provides a kit of parts comprising a pharmaceuticalcomposition of invention, and an administration vehicle including, butnot limited to, capsules for oral administration, inhalers for lungadministration and injectable solutions for intravenous administration.

According to a fourth aspect of the invention, there is provided amethod of treatment of a blood clotting disease where the methodcomprises administration of a composition of the present invention to apatient in need thereof. This aspect of the invention therefore alsoincludes uses of such compositions in said methods.

Blood clotting diseases may be characterized by a loss of function of ablood clotting factor, or the generation of auto-antibodies. Examples ofblood clotting diseases includes hemophilia A and acquired hemophilia A.

As used herein, the term “treatment” includes any regime that canbenefit a human or a non-human animal. The treatment of “non-humananimals” extends to the treatment of domestic animals, including horsesand companion animals (e.g. cats and dogs) and farm/agricultural animalsincluding members of the ovine, caprine, porcine, bovine and equinefamilies. The treatment may be in respect of any existing condition ordisorder, or may be prophylactic (preventive treatment). The treatmentmay be of an inherited or an acquired disease. The treatment may be ofan acute or chronic condition.

Nucleophilic groups on proteins, including antibodies, which can be usedto conjugate polymer in accordance with an aspect of the presentinvention include, but are not limited to: (i) N-terminal amine groups,(ii) side chain amine groups, e.g. lysine, (iii) side chain thiolgroups, e.g. cysteine, and (iv) sugar hydroxyl or amino groups where theprotein is glycosylated. Amine, thiol, and hydroxyl groups arenucleophilic and capable of reacting to form covalent bonds withelectrophilic groups on linker moieties and linker reagents attached tothe polymer including: (i) active esters such as NHS esters. HOBtesters, haloformates, and acid halides; (ii) alkyl and benzyl halidessuch as haloacetamides; (iii) aldehydes, ketones, carboxyl, andmaleimide groups. Many proteins, including antibodies, have cysteinethiol groups which can potentially be used for conjugation. Manycysteine residues are in the form of reducible interchain disulfides,i.e. cysteine bridges. Cysteine residues in the form of disulfides aregenerally not available to react with reagents such as maleimide.Cysteine residues may also be free or unpaired. However, free cysteineresidues are frequently found to be “capped” by one or more reagents invarious media and are also not available for conjugation. Cysteineresidues may be made reactive for conjugation with linker reagents suchas maleimide by treatment with a reducing agent such as DTT(dithiothreitol) or tricarbonylethylphosphine (TCEP), such that theprotein is fully or partially reduced. Each cysteine bridge will thusform, theoretically, two reactive thiol nucleophiles. In the case offree cysteine, one thiol nucleophile is formed by reduction. Dependingcm the conditions employed, reduction by TCEP or DTT can result in theloss of proper protein folding with concomitant loss of activity.However, activity may be recovered by allowing protein refolding underthe appropriate conditions.

Additional nucleophilic groups can be introduced into antibodies throughmodification of lysine residues, e.g., by reacting lysine residues with2-iminothiolane (Traut's reagent), resulting in conversion of an amineinto a thiol. Reactive thiol groups may be introduced into a protein byintroducing one, two, three, four, or more cysteine residues (e.g., bypreparing variant comprising one or more non-native cysteine amino acidresidues).

IV. Examples Synthesis of Initiators Example 1. Preparation of 3-Arm“Snap-On” Initiator

A TFA/amine salt initiator (Compound B) having the structure below wassynthesized as follows.

First, a BOC protected 3-arm initiator. Compound A, having the followingstructure:

was prepared as follows: into a 25 mL round bottom flask under nitrogenwas placed tert-butyl 2-[2-(2-aminoethoxy)ethoxy]ethylcarbamate (66 mg,0.26 mmol, 1.2 equiv) and(2,2,2-Tri(2-bromo-2-methyl-propionyloxymethyl)-ethoxy)-acetic acid(prepared as described in PCT/US2012/060301 for Product 4.5, which isincorporated herein by reference) (142 mg, 0.22 mmol, 1.0 equiv)followed by N,N-dimethylformamide (2 mL) and thenN,N-diisopropylethylamine (0.19 mL, 1.1 mmol, 5.0 equiv). The flask wascooled to 0° C. using an ice bath. To this was added propylphosphonicanhydride solution (50 wt. % in ethyl acetate, 0.16 mL, 0.26 mmol, 1.2equiv) over 1 minute. The reaction was warmed to room temperature andstirred for 1.5 hours. The reaction was quenched by adding water, thenpartitioned using water and ethyl acetate. The organic layer wasseparated and the aqueous layer extracted with ethyl acetate. Thecombined organic layers were washed with water, saturated aqueous sodiumbicarbonate, water, 0.5 M aqueous citric acid, water, then dried (sodiumsulfate), filtered and concentrated under vacuum. The residue wasapplied onto a silica gel column (60 mL) and eluted with 70% ethylacetate with 30% hexanes. The tubes containing product was pooled andconcentrated under vacuum, which resulted in 150 mg (0.17 mmol, 77%) ofCompound A.

1H NMR (400 MHz CDCl3): δ=Need to put in data 1.44 (s, 9H, OCCH3), 1.96(s, 18H, CC(CH3)2Br), 3.31 (q, J=4.8 Hz, 2H, OCNHCH2CH20), 3.5-3.6 (m,12H), 3.99 (s, 2H, OCH2C), 4.32 (s, 6H, CCH20C=0), 5.0 (br s, 1H,CH2NHC=00), 6.8 (br s, 1H. CH2NHC═OC), LC-MS (ES, m/z): [M+H]+ Calcd forC30H51Br3N2O12+H=871.1. Found 871.8.

Compound A was de-protected to yield Compound B as follows: into a 20 mLround bottom under nitrogen was added Compound A (120 mg, 0.14 mmol, 1equiv), dichloromethane (2 mL) followed by trifluoroacetic acid (2 mL,26.9 mmol, 192 equiv). The reaction stirred at room temperature for 30minutes. The reaction was diluted using hexanes dichloromethane (20 mL)and concentrated under a vacuum. The reaction was diluted using hexanes(50 mL) and concentrated under vacuum (twice), which resulted in 2.2 g(2.73 mmol, (with residual dichloromethane)) of compound B.

1H NMR (400 MHz CDCl3): δ=1.94 (s, 18H, CC(CH3)2Br), 3.2 (br, 2H,OCNHCH2CH20), 3.5-3.8 (m, 12H), 3.99 (s, 2H, OCH2C), 4.34 (s, 6H,CCH20C=0), 7.11 (br t, 1H, CH2NHC=0), 7.99 (br, 3H, NH3+).

LC-MS (ES, m/z): [M+H]+ Calcd for C25H43Br3N2O10+H=771.1. Found 771.6.

Example 2. Preparation of 6-Arm “Snap-On” Initiator

A TFA/amine salt initiator (Compound Fl) having the structure below wassynthesized

As a first step in preparing Fl, Compound C, having the followingstructure, was synthesized:

into a 100 mL round bottom under nitrogen and using a reflux condenserwas added 1-tosyl-11-(3,4,7-triaza-4,6,10-triphenyl-adamantan-1-ylmethoxy)-3,6,9-trioxaundecane (prepared as described inPCT/US2012/060301 for Product 2.2) (4.0 g, 5.5 mmol, 1.0 equiv),di(tert-butyl) imidodicarbonate (1.43 g, 6.6 mmol, 1.2 equiv), potassiumcarbonate (1.9 g, 13.7 mmol, 2.5 equiv), potassium iodide (0.137 g, 0.82mmol, 0.15 equiv) followed by acetonitrile (25 mL). The reaction wasstirred at room temperature for 5 minutes followed by stirring at 60° C.for 30 hours. The reaction was quenched by adding water (25 mL) andtert-butyl methyl ether (125 mL). The organic layer was separated andthe aqueous layer extracted with tert-butyl methyl ether (75 mL). Thecombined organic layers were washed with saturated aqueous sodiumchloride (20 mL), then dried (sodium sulfate), filtered and concentratedunder vacuum. The residue was applied onto a silica gel column (195 g,6.5 cm×12 cm) and eluted with 20% tert-butyl methyl ether in 80% hexanesup to 100% tert-butyl methyl ether. The tubes containing product werepooled and concentrated under vacuum, which resulted in 3.7 g (4.79mmol, 87%) of Compound C.

1H NMR (400 MHz DMSO-d6): δ=1.42 (s, 18H, C[C£CH3J3]2), 2.72 (s, 2H,CCH2N, isomer), 2.88 (s, 2H, CCH2N, isomer), 3.2-3.6 (m, 20H), 5.25 (s,2H, NCHPh, isomer), 5.70 (s, 1H, NCHPh, isomer), 7.3-7.8 (m, 15H,phenyl).

Next Compound D having the following structure was synthesized:

into a 500 mL round bottom under nitrogen and using a reflux condenserwas added Compound C (2.7 g, 0.3.49 mmol, 1.0 equiv), lithium hydroxidemonohydrate (0.73 g, 17.5 mmol, 5 equiv), tetrahydrofuran (20 mL),methanol (8 mL) followed by water (8 mL). The reaction was stirred at60° C. for 6 hours. The reaction was concentrated under vacuum and thenpartitioned by adding water (75 mL) and ethyl acetate (100 mL). Theorganic layer was separated and the aqueous layer extracted with ethylacetate (50 mL). The combined organic layers were washed with saturatedaqueous sodium chloride (30 mL), then dried (sodium sulfate), filteredand concentrated under vacuum. The residue was applied onto a silica gelcolumn (110 g, 5.5 cm×10.5 cm) and eluted with 50% hexanes in 50%tert-butyl methyl ether up to 100% ten-butyl methyl ether. The tubescontaining product were pooled and concentrated under vacuum, whichresulted in 1.38 g (2.05 mmol, 59%) of Compound D.

1H NMR (400 MHz DMSO-d6): δ=1.36 (s, 9H, CrC(CH3)312), 2.72 (s, 2H,CCH2N, isomer), 2.88 (s, 2H, CCH2N, isomer), 3.1-3.4 (m, 20H), 5.25 (s,2H, NCHPh (isomer)), 5.70 (s, 1H, NCHPh (isomer)), 6.73 (t, J=6.0 Hz,1H, 0=CNHCH2), 7.3-7.7 (m, 15H, phenyl).

The next step in preparing Fl was the synthesis of Compound E which hasthe following structure:

into a 100 mL round bottom was added Compound D (2.96 g, 4.4 mmol, 1.0equiv), diethyl ether (20 mL) followed by water (16 mL). The flask wascooled to 0° C. using an ice bath. To this was added hydrobromic acidsolution (48 wt % in water) (1.64 mL, 14.5 mmol, 3.3 equiv). Thereaction was stirred at 0° C. rapidly for 1 hour. The organic layer wasseparated and the aqueous layer was returned to the reaction flask at 0°C. where diethyl ether (20 mL) was added and the stirring continued for15 minutes. The organic layer was separated again and the aqueous layerwas returned to the reaction flask at 0° C. where diethyl ether (20 mL)was added and the stirring continued for 10 minutes. The organic layerwas separated and the aqueous layer was pH adjusted to 4.5 by additionof IM aqueous sodium hydroxide. The water was removed by azeotropingwith acetonitrile under a vacuum, which resulted in 2.5 g (3.85 mmol,87%) of Compound E as a white solid.

1H NMR (400 MHz DMSO-d6): δ=1.36 (s, 9H, OC(CH3)3), 3.05-3.58 (m, 24H,CH2), 6.8 (t, 1H, 0=CNHCH2), 8.0 (br s, 9H, CH2NH2*HBr).

LC-MS (ES, m/z): [M+H]+ Calcd for C18H40N4O6+H=409.3. Found 409.6.

The next step in preparation of Compound F1 was the synthesis ofCompound F, which has the following structure:

into a 200 mL round bottom flask under nitrogen was placed bis2,2-[(2-bromoisobutyryl)hydroxymethyl]propionic acid (prepared asdescribed in example 7 of U.S. patent application Ser. No. 13/641,342,which is incorporated herein by reference) (2.32 g, 5.37 mmol, 3.3equiv) and Compound E (1.06 g, 1.63 mmol, 1.0 equiv) followed bydimethylformamide (15 mL) then diisopropylethylamine (3.4 mL, 19.5 mmol,12 equiv). To this was added propylphosphonic anhydride solution (50 wt.% in ethyl acetate, 3.7 mL, 5.87 mmol, 3.5 equiv). The reaction wasstirred for 60 minutes. The reaction was quenched by adding water (1 mL)and loaded onto a preparatory HPLC column and eluted with 50%acetonitrile in water (with 0.1% trifluoroacetic acid) up to 95%acetonitrile (with 0.1% trifluoroacetic acid). The tubes containingproduct were pooled, concentrated under vacuum, frozen and placed on alyophilizer. This resulted in 640 mgs (0.39 mmol, 24%) Compound F.

Lastly, the tBOC protective group was removed to provide the finalinitiator. Fl, which has the structure shown above. Into a 50 mL roundbottom was added Product 174-44 (600 mg, 0.36 mmol) and dichloromethane(3.6 mL). The flask was cooled to 0° C. using an ice bath. To this wasadded trifluoroacetic acid (3.6 mL). The reaction was stirred at roomtemperature for 45 minutes. The reaction was diluted with hexanes andthen concentrated under a vacuum. The reaction was diluted using hexanesand concentrated under a vacuum. The residue was dissolved usingacetonitrile (3 mL), diluted with water (1.5 mL), frozen and placed on alyophilizer. This resulted in 537 mgs (0.32 mmol, 89%) of Compound Fl asan oil.

Example 3. Preparation of 9-Arm “Snap-On” Initiator Compound L

A TFA/amine salt initiator (Compound L) having the structure below wassynthesized as follows.

First, Compound K, having the following structure, was synthesized:

into a 200 mL round bottom flask under nitrogen was placed Compound J(1.9 g, 2.67 mmol, 3.3 equiv)

and Compound E (0.525 g, 0.81 mmol, 1.0 equiv) (see above) followed bydimethylformamide (10 mL) then diisopropylethylamine (2.5 mL, 14.6 mmol,18 equiv). The flask was cooled to 0° C. using an ice bath. To this wasadded propylphosphonic anhydride solution (50 wt. % in ethyl acetate,2.5 mL, 4.04 mmol, 5 equiv) over 6 minutes.

The reaction was warmed to room temperature and stirred for 15 minutes.The reaction was quenched by adding water (20 mL), saturated aqueoussodium bicarbonate (20 mL) and ethyl acetate (100 mL). The organic layerwas separated and the aqueous layer extracted with ethyl acetate (75mL). The combined organic layers were washed with saturated aqueoussodium bicarbonate (30 mL), 0.5 M aqueous citric acid (40 mL), water (25mL), and saturated aqueous sodium chloride (40 mL), then dried (sodiumsulfate), filtered and concentrated under vacuum. The residue which wasused without further purification resulted in 2.0 g (0.80 mmol, 99%) ofCompound K.

1H NMR (400 MHz DMSO-d6): δ=1.36 (s, 9H, OCCH3), 1.90 (s, 54H,CC(CH3)2Br), 2.31 (t, J=7.2 Hz, 6H, CCH2CH2NH), 2.98 (d, J=5.6 Hz, 6H,CCH2NH), 3.04 (q, J=6.0 Hz, 2H, OCH2CH2NH), 3.18 (s, 2H, OCH2C),3.3-3.37 (m, 8H, CH2), 3.47-3.55 (m, 12H, CH2), 3.58 (s, 6H, OCH2C),3.87 (s, 6H, 0=CCH20), 4.27 (s, 18H, CCH20C=0), 6.74 (br t, 1H,CH2NHC=0), 7.69 (t, J=6.8 Hz, 3H, CH2NHC=0), 7.84 (t, J=6.0 Hz, 3H,CH2NHC=0).

LC-MS (ES, m/z): [(M+2H-boc)/2]+ Calcd for(C84H136Br9N7033+2H-Boc)/2=1196.6. Found 1196.6.

Next, Compound L was synthesized as follows: into a 100 mL round bottomunder nitrogen was added Compound K (2.0 g, 0.8 mmol), dichloromethane(10 mL) followed by trifluoroacetic acid (5 mL). The reaction wasstirred at room temperature for 30 minutes. The reaction wasconcentrated under a vacuum. The reaction was diluted usingdichloromethane (10 mL) and concentrated under a vacuum. The residue wasdissolved using acetonitrile (10 mL), filtered through a syringe filter(Acrodisc CR25, PN 4225T) and loaded onto a preparatory HPLC column andeluted with 60% acetonitrile in water (with 0.1% trifluoroacetic acid)up to 98% acetonitrile (with 0.1% trifluoroacetic acid). The tubescontaining product were pooled, concentrated under vacuum, frozen andplaced on a lyophilizer. This resulted in 990 mgs (0.4 mmol, 50% over 2steps) Compound L as a white powder.

1H NMR (400 MHz DMSO-d6): δ=1.90 (s, 54H, CC(CH3)2Br), 2.31 (t, J=7.2Hz, 6H, CCH2CH2NH), 2.97-3.0 (m, 8H, CCH2NH and OCH2CH2NH), 3.17 (s, 2H,OCH2C), 3.3 (q, 6H, CH2CH2NHC=0), 3.4-3.59 (m, 20H, CH2)·3.87 (s, 6H,0=CCH20), 4.27 (s, 18H, CCH20C=0), 7.69-7.84 (m, 9H, both CH2NHC=Q andNH3+).

LC-MS (ES, m/z): [(M+2H)/2]+ Calcd for (C84H136Br9N7033+2H)/2=1196.6.Found 1197.4.

Example 4. Preparation of Longer Spacer 9-Arm Initiator SnapOn InitiatorCompound O

A TFA/amine salt initiator (Compound O) having the structure below wassynthesized as follows:

First, Compound M, having the following structure, was synthesized:

into a 20 mL vial was placed Compound L (410 mg, 0.164 mmol, 1.0 equiv)(see above) and fl/p/zfl-i-butyloxycarbonylamino-omegfl-carboxyocta(ethylene glycol) (97.5 mg, 0.18 mmol, 1.1 equiv) followed byN,N-dimethylformamide (2 mL) then N,N-diisopropylethylamine (0.171 mL,0.982 mmol, 6 equiv). The flask was cooled to 0° C. using an ice bath.To this was added propylphosphonic anhydride solution (50 wt. % in ethylacetate, 0.205 mL, 0.327 mmol, 2 equiv) over −1 minutes. The reactionwas warmed to room temperature and stirred for 30 minutes. The reactionwas quenched by adding water (10 mL), saturated aqueous sodiumbicarbonate (10 mL) and ethyl acetate (40 mL). The organic layer wasseparated and the aqueous layer extracted with ethyl acetate (25 mL).The combined organic layers were washed with saturated aqueous sodiumbicarbonate (10 mL), 0.5 M aqueous citric acid (10 mL), water (10 mL),and saturated aqueous sodium chloride (10 mL), then dried (sodiumsulfate), filtered and concentrated under vacuum. The residue which wasused without further purification resulted in 0.5 g (0.172 mmol, 105%)of Compound M.

LC-MS (ES, m/z): [(M+2H-boc)/2]+ Calcd for(C103H173Br9N8O42+2H-Boc)/2=1408.2. Found 1408.9.

Into a 100 mL round bottom under nitrogen was added Compound M (0.5 g),dichloromethane (4 mL) followed by trifluoroacetic acid (3 mL). Thereaction stirred at room temperature for 15 minutes. The reaction wasconcentrated under a vacuum. The residue was dissolved usingacetonitrile (3 mL), filtered through a syringe filter (Acrodisc CR25,PN 4225T) and loaded onto a preparatory HPLC column and eluted with 50%acetonitrile (with 0.1% trifluoroacetic acid) in 50% water (with 0.1%trifluoroacetic acid) up to 90% acetonitrile (with 0.1% trifluoroaceticacid). The tubes containing product were pooled, concentrated undervacuum, frozen and placed on a lyophilizer. This resulted in 101 mgs(21% over 2 steps) Compound O.

1H NMR (400 MHz DMSO-d6): δ=1.90 (s, 54H, CC(CH3)2Br), 2.3 (br t, 8H,CCH2CH2NH and CH2CH2C=0), 3.0 (m, 8H, CCH2NH and OCH2CH2NH), 3.1-3.6 (m,64H, OCH2C), 3.87 (s, 6H, 0=CCH20), 4.27 (s, 18H, CCH20C=0), 7.6-7.8 (m,10H, both CH2NHC=Q and NH3+).

LC-MS (ES, m/z): [(M+2H)/2]+ Calcd for (C98H165Br9N8O40+2H)/2=1408.2.Found 1408.3.

Example 5. Preparation of Longer Spacer 9-Arm “Snap-On” InitiatorCompound P

A TFA/amine salt initiator (Compound P) having the structure below wassynthesized as follows:

into a 20 mL vial was placed Compound L (430 mg, 0.172 mmol, 1.0 equiv)(see above) and alpha-t-Butyloxycarbonylamino-omega-carboxydodeca(ethylene glycol) (154 mg, 0.215 mmol, 1.25 equiv) followed byN,N-dimethylformamide (2 mL) then N,N-diisopropylethylamine (0.18 mL,1.03 mmol, 6 equiv). The flask was cooled to 0° C. using an ice bath. Tothis was added propylphosphonic anhydride solution (50 wt. % in ethylacetate, 0.215 mL, 0.343 mmol, 2 equiv) over 1 minute. The reaction waswarmed to room temperature and stirred for 30 minutes. The reaction wasquenched by adding water, saturated aqueous sodium bicarbonate and ethylacetate. The organic layer was separated and the aqueous layer extractedwith ethyl acetate. The combined organic layers were washed withsaturated aqueous sodium bicarbonate, 0.5 M aqueous citric acid, water,and saturated aqueous sodium chloride, then dried (sodium sulfate),filtered and concentrated under vacuum. The residue which was usedwithout further purification resulted in 0.6 g (0.194 mmol) of CompoundN, shown below.

LC-MS (ES, m/z): [(M+2H-boc)/2]+ Calcd for(ClllH189Br9N8046+2H-Boc)/2=1496.3. Found 1497.2.

into a 100 mL round bottom under nitrogen was added Compound N (0.6 g),dichloromethane (4 mL) followed by trifluoroacetic acid (3 mL). Thereaction stirred at room temperature for 15 minutes. The reaction wasconcentrated under a vacuum. The residue was dissolved usingacetonitrile (3 mL), filtered through a syringe filter (Acrodisc CR25,PN 4225T) and loaded onto a preparatory HPLC column and eluted with 50%acetonitrile (with 0.1% trifluoroacetic acid) in 50% water (with 0.1%trifluoroacetic acid) up to 90% acetonitrile (with 0.1% trifluoroaceticacid). The tubes containing product were pooled, concentrated undervacuum, frozen and placed on a lyophilizer. This resulted in 200 mgs(0.064 mmol, 37% over 2 steps) Compound P.

1H NMR (400 MHz DMSO-d6): δ=1.90 (s, 54H, CC(CH3)2Br), 2.3 (br t, 8H,CCH2CH2NH and CH2CH2C=0), 3.0 (m, 8H, CCH2NH and OCH2CH2NH), 3.1-3.6 (m,84H, OCH2C), 3.87 (s, 6H, 0=CCH20), 4.27 (s, 18H, CCH20C=0), 7.6-7.8 (m,10H, both CH2NHC=Q and NH3+).

LC-MS (ES, m/z): [(M+2H)/2]+ Calcd for (C106H181Br9N8O44+2H)/2=1496.3.Found 1496.6.

Synthesis of Polymers

Example 6. Preparation of Zwitterionic Polymers

Initiator is typically prepared as a stock solution in DMF of about 100mg/mL. The initiator and the ligand (2,2′-bipyridyl) were introducedinto a Schlenk tube. The resultant solution was cooled to −78° C. usinga dry ice/acetone mixture, and was degassed under vacuum for lOmin. Thetube was refilled under Argon and the catalyst (CuBr unless otherwiseindicated), kept under Argon, was introduced into the Schlenck tube (theMolar ratio of atom bromine on the initiator/catalyst (CuBr)-ligand waskept at 1/1/2). The solution became dark brown immediately. The Schlenktube was sealed and immediately purged by applying a short cyclevacuum/Argon. A solution of HEMA-PC was prepared by mixing a definedquantity of monomer, prepared in a glovebox kept under nitrogen, with200 proof degassed ethanol. The monomer solution was added drop wiseinto the Schlenk tube (via canula) (and homogenized by light stirring:unnecessary). The temperature was maintained at −78° C. A thoroughvacuum was applied to the reaction mixture for at least 10 to 15 min,until bubbling from the solution ceased. The tube was then refilled withArgon and warmed to room temperature. The solution was stirred, and asthe polymerization proceeded, the solution became viscous. After 3 to 8hours or just left overnight, the reaction was quenched by directexposure to air in order to oxidize Cu (I) to Cu (II), the mixturebecame blue-green in color, and was passed through a silica column inorder to remove the copper catalyst. The collected solution wasconcentrated by rotary evaporation and the resulting mixture was eitherprecipitated with tetrahydrofuran or dialyzed against water followed byfreeze drying to yield a free-flowing white powder. Table 2 sets forthexemplary polymers made in accordance with the present invention.

TABLE 2 Theor. MW Initiator Mn Mp (kDa) Polymer ID No. (see Table 3)(kDa) (kDa) PDI 150 60 B5 150 158 1.05 [w2199] [d2191] 250 70 B4 205 2301.05 [wR2765] [dR2731] 250 80 B5 117 137 1.1 [wR3350] [dR3324] 250 90 B62.35 258 1.1 [wR3373] [dR3372] 250 100 B 242 259 1.1 [wR3460I] [wR3461M][dR3418] 250 110 F1 245 270 1.1 [wR3482I] [wR3483M] [wR3463] 500 120 F1490 557 1.1 [wR3763] [dR3662] 500 130 L 490 530 1.1 [wR3758] [dR3706]750 140 F1 586 695 1.1 [wR3764] [dR3667] 750 150 L 645 750 1.1 [wR3759][dR3707] 750 160 O 656 740 1.1 [wR3836] [dR3804] 750 170 P 785 900 1.1[wR3835] [dR3808]

TABLE 3 B

B2

B3

B4

B5

B6

F1

L

O

P

Example 7. Deprotection of Protected Maleimide

It was observed that the protected maleimide biopolymers tend to shiftthe Mp to higher values after heat de-protection when the biopolymerpowder was heated at 120° C. for 90 minutes. This makes the biopolymermanufacturing more challenging since the amount of up-shift in Mpdepended on the biopolymer (Mp, architecture, etc). An alternativemethod of deprotection is needed.

Initial experimentation was carried out in water in a sealed capillaryloop. It was demonstrated that furan was released during heatdeprotection of aqueous biopolymer solution in an oven at 120° C. Noup-shift in Mp was observed after the heat deprotection in aqueoussolution.

The procedure was also repeated for biopolymers dissolved in ethanol. Itwas confirmed that the Mp up-shift was completely eliminated when theheat deprotection was carried out in ethanol solution. Different heatingmethods such as an oven or an oil bath were tested and little differencewas found as long as the heating time and temperatures were kept thesame. The duration of heating has to be optimized to avoid biopolymerdegradation but at the same time ensuring deprotection of most of thefuran protected maleimide biopolymers. The procedure was finalized touse an ethanol solution of the biopolymers in a pressure reactor (ableto hold 70 PSI pressure).

Typical operation starts with a clean and dry glass reactor. Thebiopolymer is dissolved in ethanol to form a clear and transparentsolution. The concentration of the biopolymer is typically between 50mg/mL to 150 mg/mL, with around 100 mg/mL most often. This is a goodbalance between minimizing the ethanol to be consumed and avoiding highviscosity of the polymer solution.

The clear biopolymer solution should be transferred to a clean pressurereactor, purged with N₂ for 3 to 5 minutes, and then tightly capped. Themass of the reactor plus the biopolymer solution should be logged beforeand after the heat deprotection so any leak can be identified.

The pressure reactor containing the biopolymer (to be deprotected)solution is placed in an oven set at 120° C. for two hours. After thedeprotection, the pressure reactor is taken out of the oven and allowedto cool down. The deprotected biopolymer solution can be purified bysolvent precipitation, spray-drying, or lyophilization.

Preparation of Conjugatable Polymers

Example 8. Preparation of Maleimide Conjugatable 3-Arm Polymer

A maleimide conjugatable polymer (B3) having the following structure wasprepared as follows:

into a 20 mL vial was placed Polymer ID No. 100 (Table 2) (280 mg,0.00123 mmol, 1.0 equiv) and dissolved using water (2 mL). To this wasadded 0.5 M aqueous sodium phosphate dibasic (0.2 mL). In a separatevial was dissolved 3-maleimidopropionic acid, NHS ester (1.5 mg, 0.00548mmol, 4.5 equiv) in tetrahydrofuran (0.6 mL). The NHS ester solution wasadded to the polymer solution over 2 minutes at room temperature and theresulting solution was stirred for 75 minutes. The reaction was dilutedwith 4:1 water:tertahydrofuran (4 mL) and placed into a Amiconcentrifuge membrane dialysis tube (30,000 mwco) and the tube placed intocentrifuge (rpm 3000) for 30 minutes. The filtrate is removed foranalysis while the retentate is diluted and mixed with 4:1water:tertahydrofuran (6 mL) and the tube placed into centrifuge (rpm3000) for 30 minutes. The filtrate is removed for analysis while theretentate is diluted and mixed with water (8 mL), placed into centrifuge(rpm 3000) for 30 minutes. The filtrate is removed for analysis whilethe retentate is diluted and mixed with water (8 mL). The centrifugeprocedure repeated 3 more times, after which the retentate is removedand placed into a vial. The Amicon membrane tube was rinsed with water(2×˜2 mL) and this combined with the retentate, which was frozen andplaced on a lyophilizer. This resulted in 262 mgs (93%) B3 as a whitepowder.

Example 9. Preparation of Maleimide 6-Arm Conjugatable Polymer

A maleimide conjugatable 6-arm polymer (F4) having the followingstructure was synthesized as follows:

into a 20 mL vial was placed Polymer ID No. 110 (Table 2) (502 mg,0.00205 mmol, 1.0 equiv) and dissolved using water (4 mL). To this wasadded 0.5 M aqueous sodium phosphate dibasic (0.4 mL). In a separatevial was dissolved 3-maleimidopropionic acid. NHS ester (2.45 mg, 0.0092mmol, 4.5 equiv) in tetrahydrofuran (1 mL). The NHS ester solution wasadded to the polymer solution over 2 minutes at room temperature and theresulting solution was stirred for 100 minutes. The reaction was dilutedwith 4:1 water:tertahydrofuran (4 mL) and placed evenly into 2 Amiconcentrifuge membrane dialysis tubes (30,000 mwco) and the tubes placedinto centrifuge (rpm 3000) for 30 minutes. The filtrate is removed foranalysis while the retentate is diluted and mixed with 4:1water:tertahydrofuran (6 mL) and the tubes placed into centrifuge (rpm3000) for 30 minutes. The filtrate is removed for analysis while theretentate is diluted and mixed with water (8 mL each), placed intocentrifuge (rpm 3000) for 30 minutes. The filtrate is removed foranalysis while the retentate is diluted and mixed with water (8mL/tube). The centrifuge procedure repeated 3 more times, after whichthe retentate is removed and placed into a vial. The Amicon membranetubes were rinsed with water (2×˜2 mL each tube) and this combined withthe retentate was frozen and placed on a lyophilizer. This resulted in459 mgs (91%) Polymer F4 as a white powder.

Example 10. Preparation of 6-Arm Conjugatable Polymer

A maleimide conjugatable 6-arm polymer (S) having the followingstructure was synthesized as follows:

into a 20 mL vial was placed Polymer ID No. 120 (Table 2) (500 mg,0.00091 mmol, 1.0 equiv) and dissolved using ethanol (4 mL) afterstirring for 10 minutes. To this was added a 1% solution of4-methylmorpholine in acetonitrile (0.030 mL, 0.00273 mmol, 3 equiv). Ina separate vial was dissolved Product 176-55 (2.65 mg, 0.00455 mmol, 5equiv) in acetonitrile (1 mL) and this solution was added to the polymersolution over ˜1 minute at room temperature. An additional aliquot ofacetonitrile (1 mL) was added and the resulting solution was stirred for18 hours. The reaction was diluted with 0.1% aqueous trifluoroaceticacid (2 mL) (pH˜6) followed by water (−14 mL), filtered through asyringe filter (Acrodisc Supor, PN 4612) and placed evenly into 3 Amiconcentrifuge membrane dialysis tubes (30,000 mwco). The tubes were dilutedand mixed with water (˜5 mL each), placed into centrifuge (rpm 3000) for30 minutes. The filtrate is removed for analysis while the retentate isdiluted and mixed with water (−10 mL/tube). The centrifuge procedurerepeated 5 more times, after which the retentate is removed and placedinto a vial. The Amicon membrane tubes were rinsed with water (2×˜2 mLeach tube) and this combined with the retentate. The retentate solutionwas filtered through a syringe filter (Acrodisc Supor. PN 4612), frozenand placed on a lyophilizer. This resulted in 469 mgs (0.00085 mmol,93%) Polymer S as a white powder.

Example 11. Preparation of Maleimide 9-Arm Conjugatable Polymer

A maleimide conjugatable 9-arm polymer (Q) having the followingstructure was synthesized as follows:

conjugatable polymer Q was prepared as follows: into a 20 mL vial wasplaced Polymer ID No. 160 (Table 2) (540 mg, 0.0007 mmol, 1.0 equiv) anddissolved using water (4 mL). To this was added 0.5 M aqueous sodiumphosphate dibasic (0.4 mL). In a separate vial was dissolved3-maleimidopropionic acid, NHS ester (0.93 mg, 0.0035 mmol, 5 equiv) intetrahydrofuran (1 mL). The NHS ester solution was added to the polymersolution over −2 minutes at room temperature and the resulting solutionwas stirred for 30 minutes. The reaction was diluted with water (−15mL), filtered through a syringe filter (Acrodisc Super, PN 4612) andplaced evenly into 3 Amicon centrifuge membrane dialysis tubes (30,000mwco). The tubes were diluted and mixed with water (5 mL each), placedinto centrifuge (rpm 3000) for 30 minutes. The filtrate is removed foranalysis while the retentate is diluted and mixed with water (−10mL/tube). The centrifuge procedure repeated 5 more times, after whichthe retentate is removed and placed into a vial. The Amicon membranetubes were rinsed with water (2×−2 mL each tube) and this combined withthe retentate. The retentate solution was filtered through a syringefilter (Acrodisc Super, PN 4612), frozen and placed on a lyophilizer.This resulted in 508 mgs (94%) Polymer Q as a white powder.

Example 12. Preparation of Maleimide 9-Arm Conjugatable Polymer

A maleimide conjugatable 9-arm polymer (R) having the followingstructure was synthesized as follows:

It was prepared using the same techniques as describe for Conjugatablepolymer Q.

Example 13. Preparation of Wild-Type Factor VIII for Conjugation

Mammalian expressed wild-type (FVIII-WT) is known to have all cysteineresidues either oxidized to form disulfide linkages or, in the case ofthe free cysteines present in the B domain, blocked (capped) bymetabolites from the media that prevent unpaired free cysteines frombeing available for conjugation using thiol-reactive polymers containingreactive groups such as maleimide or iodoacetamide. These cappingmoieties can be removed using reducing agents such as TCEP or DTTfollowed by removal of the reducing agent and protein refolding.

FVIII-WT was formulated into 50 mM MOPS pH7, IOmM CaCl₂, 200 mM NaCl, 1%sucrose and 0.01% Tween80 at a concentration of 0.5 mg/mL. A 150× molarexcess equivalent of TCEP solution was added and incubated at 4° C. for1 hour. A desalting column of Sephadex G25 was used for TCEP removal.The G25 column was equilibrated with the formulation buffer and the TCEPreduced sample was loaded, and fractions collected were analyzed bySDS-PAGE. The fractions containing protein were pooled and incubated at4° C. overnight to allow protein refolding (regeneration of disulfidepairs by oxidation), while unpaired cysteine remained in free sulfhydrylform (decapped). Alternatively, the TCEP removal was accomplished usingan anion exchange (e.g. Q Sepharose FF) column where the TCEP reducedsample was diluted to lower the salt concentration and then loaded ontothe QFF column, followed by a washing step using the low salt MOPSbuffer and elution with a step gradient of NaCl. Under these conditions,the protein eluted at around 300 mM NaCl. The protein fractions werepooled for conjugation as described below. The ion exchange method forTCEP removal is preferred over the desalting column approach as it ismore amenable to scale up.

The analysis of the TCEP treated form by SDS-PAGE analysis showedpredominantly two bands: (1) a higher MW band migrating at around 180kDa which represents the heavy chain plus B domain (HC-BD); and (2) alower MW band migrating at about 80 kDa which represents the light chain(LC). The sample was also analyzed by gel filtration using a Superose 6column. The column was equilibrated in 20 mM Tris pH7.5, lOmM CaCl₂, 200mM NaCl, 10% ethanol, 1% sucrose and 0.001% Tween80 followed byinjecting different FVIII samples including: (1) TCEP treated andrefolded FVIII-WT; and (2) original FVIII-WT for comparison. The elutionprofile of both samples at 280 nm each showed a predominant single peakat the expected retention time.

Example 14. Conjugation of FVIII-WT to High Molecular WeightZwitterionic Polymers

The unveiled free cysteine thiol in the TCEP treated form of FVIII-WTwas used for conjugation to a variety of maleimide and iodo-acetamidefunctionalized polymers from above, varying in molecular weight,architecture, and linker length as shown in Table 4. The conjugationreaction mixtures contained FVIII-WT protein at about 0.5 mg/mL in 50 mMMOPS pH7, lOmM CaCl₂, 200 mM NaCl, 0.01% Tween80 and 5-100× molar excessof the maleimide polymer dissolved in 20 mM Tris pH8, 200 mM NaCl, lOmMCaCl₂, and 0.01% Tween80. The reactions proceeded at 4° C. overnightfollowed by analysis of the conjugation efficiency by SDS-PAGE underboth non-reducing and reducing conditions. The results showed thedisappearance of a single heavy chain-B domain (HC-BD) band but nottruncated forms of the domain and the concomitant appearance of a highmolecular weight band at the top of the gel indicating the presence ofnewly formed conjugate. The conjugation efficiency (calculated as thepercentage of the remaining HC-B domain band compared with the nopolymer control) of each reaction is shown in Table 4.

TABLE 4 Conjugation of funetionalized Polymers to WT FVIII Polymer Init.“Snap on” Mp Molar Conj. No. (Table 2) moiety PDI (kD) Excess (x)Effic.* 1 B4 None—heat 1.041 226.9  5x − deprotect 2 B5 None—heat 1.089240.5  10x + to + + deprotect 3 B6 None—heat 1.085 255.4  10x + to + +deprotect 4 B 

1.072 269.3  10x + + 5 F1

1.075 266.5  10x + 6 F1

1.088 552.8  50x + to + + 7 F1

1.102 696.9  50x + to + + 8 L 

1.082 531.7  50x + to + + 9 L 

1.098 768.3  50x + to + + 10 F1

1.088 552.8  50x + 11 F1

1.102 696.9  50x + 12 L 

1.082 531.7  50x + 13 L 

1.098 768.3  50x + 14 B5 Noce—heat 1.089 240.5   50x, + + + + deprotect100x + + + + 15 F1

1.102 696.9 100x + + + + + + + 16 L 

1.098 768.3  50x 100x + + + + + + + 17 P 

1.095 769.3  50x 100x + + + + + + + 18 O 

1.119 749.5 100x + + + + + + + * + + + + = excellent; + + + = good; + += fair; + = low; − = none or undetectable.Table 5 below shows activity of conjugates formed from 9-branch hema-PCpolymers from initiator O or P from Table 3.

QC3228 (Conjugate concentration by OD280 peak area of the conjugate)Specific Cone, Activity activity Mn Mp Mw Polymer (mg/ml) (IU/mg)(IU/mg) QC# Note PDI (kD) (kD) (kD) used Initiator Conjugate nameQC3228_R4370 1.81 3260.3 1801.3 QC3191 WT-OG1706- 1.070 1299 1296 1389OG1466 O conjugate QC3228_R3961 3.70 8758.4 2367.1 QC3191 WT-OG1502-1.052 1175 1171 1236 OG1465 P conjugate FVIII parent proteinsOG1297_R3971 0.2 597.5 2987.5 QC3237 WT-Original- 1.110 289 304 320stock Polymer used OG1466_R3836_mean Mean = 1.066 764 792 815OG1465_R3835_mean Mean = 1.097 676 774 741

A conjugation reaction was performed using lmg of TCEP-treated FVIII-WTand a 50× molar excess of the conjugatable polymer used in no. 17 (Table4) and the protocol described previously. A conjugation efficiencyof >90% was determined using SDS-PAGE as before. The conjugate band wasmaintained under reducing conditions.

The conjugate was purified using cation exchange chromatography usingMacroCap SP resin. The conjugation reaction was diluted lOx into 50 mMMOPS, pH7, lOmM CaCl₂, 0.01% Tween80 and loaded onto 3 mL resin packedinto a 5 mL drip column. Column flow was achieved by gravity and theunbound fraction collected. The column was chased and washed with acombined 2 l column volume (CV) of wash buffer containing 20 mM NaCl.The bound protein was then eluted with wash buffer containing varyingNaCl concentrations including 100, 150, 200, 250 and 500 mM NaCl. Atleast 5 CV of elution was collected for each NaCl concentration. Thefractions were subjected to SDS-PAGE analysis to determine at which NaClconcentration protein was eluted. Preliminary analysis indicated thatfree protein eluted at 150 mM salt, and the conjugate eluted at lOOmMsalt. This was confirmed by analytical gel filtration on a Superose 6column which gave single peaks for conjugate and free protein. Theconjugate pool was concentrated and sterile filtered using a 0.2μιηSpinX centrifuge filter to yield a final concentration (as it relates toprotein) of 2.16 mg/mL with a final process yield of 40%.

The activity of the conjugate, determined using the COAMATIC FactorFVIII assay kit, was equivalent to the FVIII-WT.

Although the foregoing invention has been described in some detail byway of illustration and example, for purposes of clarity ofunderstanding, certain changes and modifications can be practiced withinthe scope of the appended claims. In addition, each reference providedherein is incorporated by reference in its entirety for all purposes tothe same extent as if each reference was individually incorporated byreference. To the extent the content of any citation, including websiteor accession number may change with time, the version in effect at thefiling date of this application is meant. Unless otherwise apparent fromthe context any step, element, aspect, feature of embodiment can be usedin combination with any other.

What is claimed is:
 1. A compound having the following formula:

wherein X is an integer from 1 to 50 and Y is an integer from 1 to 50;and wherein Z is polymer, the polymer is synthesized with a monomerselected from the group consisting of:

wherein R7 is H or C₁₋₆ alkyl, ZW is a zwitterion, and t is 1 to
 6. 2.The compound of claim 1, wherein the zwitterion is phosphorylcholine. 3.The compound of claim 1, wherein the monomer is2-(methacryloyloxyethyl)-2′-(trimethylammoniumethyl) phosphate (MPC) or2-(acryloyloxyethyl)-2′-(trimethylammoniumethyl) phosphate.
 4. Thecompound of claim 2, wherein the monomer is2-(methacryloyloxyethyl)-2′-(trimethylammoniumethyl) phosphate (MPC). 5.The compound according to claim 1, wherein the total molecular weight ofthe compound is about 500,000 to about 1,000,000 daltons.
 6. Thecompound according to claim 2, wherein the total molecular weight of thecompound is about 650,000 to about 850,000 daltons.
 7. The compoundaccording to claim 3, wherein the total molecular weight of the compoundis about 750,000 daltons.
 8. A compound selected from the groupconsisting of:

and wherein Z is polymer, the polymer is synthesized with a monomerselected from the group consisting of:

wherein R7 is H or C₁₋₆ alkyl, ZW is a zwitterion, and t is 1 to
 6. 9. Amethod of synthesizing a polymer-functional agent conjugate comprisingcoupling a functional agent to an unreacted reactive group of thecompound of claim 1 to provide the polymer-functional agent conjugate.10. The compound of claim 8, wherein the zwitterion isphosphorylcholine.
 11. The compound of claim 10, wherein the monomer is2-(methacryloyloxyethyl)-2 ‘-(trimethylammoniumethyl) phosphate (MPC) or2-(acryloyloxyethyl)-2’-(trimethylammoniumethyl) phosphate.
 12. Thecompound of claim 10, wherein the monomer is2-(methacryloyloxyethyl)-2′-(trimethylammoniumethyl) phosphate (MPC).13. The compound according to claim 8, wherein the total molecularweight of the compound is about 500,000 to about 1,000,000 daltons. 14.The compound according to claim 11, wherein the total molecular weightof the compound is about 650,000 to about 850,000 daltons.
 15. Thecompound according to claim 12, wherein the total molecular weight ofthe compound is about 750,000 daltons.